Angiostatin Fragments and Method of Use

ABSTRACT

Fragments of an endothelial cell proliferation inhibitor and method of use therefor are provided. The endothelial proliferation inhibitor is a protein derived from plasminogen, or more specifically is an angiostatin fragment. The angiostatin fragments generally correspond to kringle structures occurring within the endothelial cell proliferation inhibitor. The endothelial cell inhibiting activity of these fragments provides a means for inhibiting angiogenesis of tumors and for treating angiogenic-mediated disease.

FIELD OF THE INVENTION

The present invention relates to endothelial inhibitors, calledangiostatin, which reversibly inhibit proliferation of endothelialcells. More particularly, the present invention relates to angiostatinproteins that can be isolated from body fluids such as blood or urine,or can be synthesized by recombinant, enzymatic or chemical methods. Theangiostatin is capable of inhibiting angiogenesis related diseases andmodulating angiogenic processes. In addition, the present inventionrelates to diagnostic assays and kits for angiostatin measurement, tohistochemical kits for localization of angiostatin, to DNA sequencescoding for angiostatin and molecular probes to monitor angiostatinbiosynthesis, to antibodies that are specific for the angiostatin, tothe development of protein agonists and antagonists to the angiostatinreceptor, to anti-angiostatin receptor-specific antibody agonists andantagonists, and to cytotoxic agents linked to angiostatin proteins.

BACKGROUND OF THE INVENTION

As used herein, the term “angiogenesis” means the generation of newblood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals undergo angiogenesis only in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The term “endothelium” means athin layer of flat epithelial cells that lines serous cavities, lymphvessels, and blood vessels.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, tumor metastasis and abnormal growth by endothelial cells andsupports the pathological damage seen in these conditions. The diversepathological disease states in which unregulated angiogenesis is presenthave been grouped together as angiogenic dependent or angiogenicassociated diseases.

The hypothesis that tumor growth is angiogenesis-dependent was firstproposed in 1971. (Folkman J., Tumor angiogenesis: Therapeuticimplications., N. Engl. Jour. Med. 285:1182 1186, 1971) In its simplestterms it states: “Once tumor ‘take’ has occurred, every increase intumor cell population must be preceded by an increase in new capillariesconverging on the tumor.” Tumor ‘take’ is currently understood toindicate a prevascular phase of tumor growth in which a population oftumor cells occupying a few cubic millimeters volume and not exceeding afew million cells, can survive on existing host microvessels. Expansionof tumor volume beyond this phase requires the induction of newcapillary blood vessels. For example, pulmonary micrometastases in theearly prevascular phase in mice would be undetectable except by highpower microscopy on histological sections.

Examples of the indirect evidence which support this concept include:

(1) The growth rate of tumors implanted in subcutaneous transparentchambers in mice is slow and linear before neovascularization, and rapidand nearly exponential after neovascularization. (Algire G H, et al.Vascular reactions of normal and malignant tumors in vivo. I. Vascularreactions of mice to wounds and to normal and neoplastic transplants. J.Natl. Cancer Inst. 6:73-85, 1945)

(2) Tumors grown in isolated perfused organs where blood vessels do notproliferate are limited to 1-2 mm³ but expand rapidly to >1000 timesthis volume when they are transplanted to mice and becomeneovascularized. (Folkman J, et al., Tumor behavior in isolated perfusedorgans: In vitro growth and metastasis of biopsy material in rabbitthyroid and canine intestinal segments. Annals of Surgery 164:491-502,1966)

(3) Tumor growth in the avascular cornea proceeds slowly and at a linearrate, but switches to exponential growth after neovascularization.(Gimbrone, M. A., Jr. et al., Tumor growth and neovascularization: Anexperimental model using the rabbit cornea. J. Natl. Cancer Institute52:41-427, 1974)

(4) Tumors suspended in the aqueous fluid of the anterior chamber of therabbit eye, remain viable, avascular and limited in size to <1 mm³. Oncethey are implanted on the iris vascular bed, they become neovascularizedand grow rapidly, reaching 16,000 times their original volume within 2weeks. (Gimbrone M A Jr., et al., Tumor dormancy in vivo by preventionof neovascularization. J. Exp. Med. 136:261-276)

(5) When tumors are implanted on the chick embryo chorioallantoicmembrane, they grow slowly during an avascular phase of >72 hours, butdo not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansionoccurs within 24 hours after the onset of neovascularization, and by day7 these vascularized tumors reach a mean diameter of 8.0+2.5 mm.(Knighton D., Avascular and vascular phases of tumor growth in the chickembryo. British J. Cancer, 35:347-356, 1977)

(6) Vascular casts of metastases in the rabbit liver revealheterogeneity in size of the metastases, but show a relatively uniformcut-off point for the size at which vascularization is present. Tumorsare generally avascular up to 1 mm in diameter, but are neovascularizedbeyond that diameter. (Lien W., et al., The blood supply of experimentalliver metastases. II. A microcirculatory study of normal and tumorvessels of the liver with the use of perfused silicone rubber. Surgery68:334-340, 1970)

(7) In transgenic mice which develop carcinomas in the beta cells of thepancreatic islets, pre-vascular hyperplastic islets are limited in sizeto <1 mm. At 6-7 weeks of age, 4-10% of the islets becomeneovascularized, and from these islets arise large vascularized tumorsof more than 1000 times the volume of the pre-vascular islets. (FolkmanJ, et al., Induction of angiogenesis during the transition fromhyperplasia to neoplasia. Nature 339:58-61, 1989)

(8) A specific antibody against VEGF (vascular endothelial growthfactor) reduces microvessel density and causes “significant or dramatic”inhibition of growth of three human tumors which rely on VEGF as theirsole mediator of angiogenesis (in nude mice). The antibody does notinhibit growth of the tumor cells in vitro. (Kim K J, et al., Inhibitionof vascular endothelial growth factor-induced angiogenesis suppressestumor growth in vivo. Nature 362:841-844, 1993)

(9) Anti-bFGF monoclonal antibody causes 70% inhibition of growth of amouse tumor which is dependent upon secretion of bFGF as its onlymediator of angiogenesis. The antibody does not inhibit growth of thetumor cells in vitro. (Hori A, et al., Suppression of solid tumor growthby immunoneutralizing monoclonal antibody against human basic fibroblastgrowth factor. Cancer Research, 51:6180-6184, 1991)

(10) Intraperitoneal injection of bFGF enhances growth of a primarytumor and its metastases by stimulating growth of capillary endothelialcells in the tumor. The tumor cells themselves lack receptors for bFGF,and bFGF is not a mitogen for the tumors cells in vitro. (Gross J L, etal. Modulation of solid tumor growth in vivo by bFGF. Proc. Amer. Assoc.Canc. Res. 31:79, 1990)

(11) A specific angiogenesis inhibitor (AGM-1470) inhibits tumor growthand metastases in vivo, but is much less active in inhibiting tumor cellproliferation in vitro. It inhibits vascular endothelial cellproliferation half-maximally at 4 logs lower concentration than itinhibits tumor cell proliferation. (Ingber D, et al., Angioinhibins:Synthetic analogues of fumagillin which inhibit angiogenesis andsuppress tumor growth. Nature, 48:555-557, 1990). There is also indirectclinical evidence that tumor growth is angiogenesis dependent.

(12) Human retinoblastomas that are metastatic to the vitreous developinto avascular spheroids which are restricted to less than 1 mm³ despitethe fact that they are viable and incorporate ³H-thymidine (when removedfrom an enucleated eye and analyzed in vitro).

(13) Carcinoma of the ovary metastasizes to the peritoneal membrane astiny avascular white seeds (1-3 mm³). These implants rarely grow largeruntil one or more of them becomes neovascularized.

(14) Intensity of neovascularization in breast cancer (Weidner N, etal., Tumor angiogenesis correlates with metastasis in invasive breastcarcinoma. N. Engl. J. Med. 324:1-8, 1991, and Weidner N, et al., Tumorangiogenesis: A new significant and independent prognostic indicator inearly-stage breast carcinoma, J Natl. Cancer Inst. 84:1875-1887, 1992)and in prostate cancer (Weidner N, Carroll P R, Flax J. Blumenfeld W,Folkman J. Tumor angiogenesis correlates with metastasis in invasiveprostate carcinoma. American Journal of Pathology, 143(2):401-409, 1993)correlates highly with risk of future metastasis.

(15) Metastasis from human cutaneous melanoma is rare prior toneovascularization. The onset of neovascularization leads to increasedthickness of the lesion and an increasing risk of metastasis.(Srivastava A, et al., The prognostic significance of tumor vascularityin intermediate thickness (0.76-4.0 mm thick) skin melanoma. Amer. J.Pathol. 133:419-423, 1988)

(16) In bladder cancer, the urinary level of an angiogenic protein,bFGF, is a more sensitive indicator of status and extent of disease thanis cytology. (Nguyen M, et al., Elevated levels of an angiogenicprotein, basic fibroblast growth factor, in urine of bladder cancerpatients. J. Natl. Cancer Inst. 85:241-242, 1993)

Thus, it is clear that angiogenesis plays a major role in the metastasisof a cancer. If this angiogenic activity could be repressed oreliminated, then the tumor, although present, would not grow. In thedisease state, prevention of angiogenesis could avert the damage causedby the invasion of the new microvascular system. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

What is needed therefore is a composition and method which can inhibitthe unwanted growth of blood vessels, especially into tumors. Alsoneeded is a method for detecting, measuring, and localizing thecomposition. The composition should be able to overcome the activity ofendogenous growth factors in premetastatic tumors and prevent theformation of the capillaries in the tumors thereby inhibiting the growthof the tumors. The composition, fragments of the composition, andantibodies specific to the composition, should also be able to modulatethe formation of capillaries in other angiogenic processes, such aswound healing and reproduction. The composition and method forinhibiting angiogenesis should preferably be non-toxic and produce fewside effects. Also needed is a method for detecting, measuring, andlocalizing the binding sites for the composition as well as sites ofbiosynthesis of the composition. The composition and fragments of thecomposition should be capable of being conjugated to other molecules forboth radioactive and non-radioactive labeling purposes

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided that are effective for modulating angiogenesis, and inhibitingunwanted angiogenesis, especially angiogenesis related to tumor growth.The present invention includes a protein, which has been named“angiostatin”, defined by its ability to overcome the angiogenicactivity of endogenous growth factors such as bFGF, in vitro, and by itamino acid sequence homology and structural similarity to an internalportion of plasminogen beginning at approximately plasminogen amino acid98. Angiostatin comprises a protein having a molecular weight of betweenapproximately 38 kilodaltons and 45 kilodaltons as determined byreducing polyacrylamide gel electrophoresis and having an amino acidsequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule (SEQ ID NO:2).

The amino acid sequence of angiostatin varies slightly between species.For example, in human angiostatin the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, fragments of human plasminogenhas similar anti-angiogenic activity as shown in a mouse tumor model. Itis to be understood that the number of amino acids in the activeangiostatin molecule may vary and all amino acid sequences that haveendothelial inhibiting activity are contemplated as being included inthe present invention.

The present invention provides methods and compositions for treatingdiseases and processes mediated by undesired and uncontrolledangiogenesis by administering to a human or animal a compositioncomprising a substantially purified angiostatin or angiostatinderivative in a dosage sufficient to inhibit angiogenesis. The presentinvention is particularly useful for treating, or for repressing thegrowth of, tumors. Administration of angiostatin to a human or animalwith prevascularized metastasized tumors will prevent the growth orexpansion of those tumors.

The present invention also encompasses DNA sequences encodingangiostatin, expression vectors containing DNA sequences encodingangiostatin, and cells containing one or more expression vectorscontaining DNA sequences encoding angiostatin. The present inventionfurther encompasses gene therapy methods whereby DNA sequences encodingangiostatin are introduced into a patient to modify in vivo angiostatinlevels.

The present invention also includes diagnostic methods and kits fordetection and measurement of angiostatin in biological fluids andtissues, and for localization of angiostatin in tissues and cells. Thediagnostic method and kit can be in any configuration well known tothose of ordinary skill in the art. The present invention also includesantibodies specific for the angiostatin molecule and portions thereof,and antibodies that inhibit the binding of antibodies specific for theangiostatin. These antibodies can be polyclonal antibodies or monoclonalantibodies. The antibodies specific for the angiostatin can be used indiagnostic kits to detect the presence and quantity of angiostatin whichis diagnostic or prognostic for the occurrence or recurrence of canceror other disease mediated by angiogenesis. Antibodies specific forangiostatin may also be administered to a human or animal to passivelyimmunize the human or animal against angiostatin, thereby reducingangiogenic inhibition.

The present invention also includes diagnostic methods and kits fordetecting the presence and quantity of antibodies that bind angiostatinin body fluids. The diagnostic method and kit can be in anyconfiguration well known to those of ordinary skill in the art.

The present invention also includes anti-angiostatin receptor-specificantibodies that bind to the angiostatin receptor and transmit theappropriate signal to the cell and act as agonists or antagonists.

The present invention also includes angiostatin protein fragments andanalogs that can be labeled isotopically or with other molecules orproteins for use in the detection and visualization of angiostatinbinding sites with techniques, including, but not limited to, positronemission tomography, autoradiography, flow cytometry, radioreceptorbinding assays, and immunohistochemistry.

These angiostatin proteins and analogs also act as agonists andantagonists at the angiostatin receptor, thereby enhancing or blockingthe biological activity of angiostatin. Such proteins are used in theisolation of the angiostatin receptor.

The present invention also includes angiostatin, angiostatin fragments,angiostatin antisera, or angiostatin receptor agonists and angiostatinreceptor antagonists linked to cytotoxic agents for therapeutic andresearch applications. Still further, angiostatin, angiostatinfragments, angiostatin antisera, angiostatin receptor agonists andangiostatin receptor antagonists are combined with pharmaceuticallyacceptable excipients, and optionally sustained-release compounds orcompositions, such as biodegradable polymers, to form therapeuticcompositions.

The present invention includes molecular probes for the ribonucleic acidand deoxyribonucleic acid involved in transcription and translation ofangiostatin. These molecular probes provide means to detect and measureangiostatin biosynthesis in tissues and cells.

Accordingly, it is an object of the present invention to provide acomposition comprising an angiostatin.

It is another object of the present invention to provide a method oftreating diseases and processes that are mediated by angiogenesis.

It is yet another object of the present invention to provide adiagnostic or prognostic method and kit for detecting the presence andamount of angiostatin in a body fluid or tissue.

It is yet another object of the present invention to provide a methodand composition for treating diseases and processes that are mediated byangiogenesis including, but not limited to, hemangioma, solid tumors,blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis,scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn'sdisease, plaque neovascularization, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, wound healing, peptic ulcer, Helicobacter relateddiseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation,menstruation, placentation, and cat scratch fever.

It is another object of the present invention to provide a compositionfor treating or repressing the growth of a cancer.

It is an object of the present invention to provide compounds thatmodulate or mimic the production or activity of enzymes that produceangiostatin in vivo or in vitro.

It is a further object of the present invention to provide angiostatinor anti-angiostatin antibodies by direct injection of angiostatin DNAinto a human or animal needing such angiostatin or anti-angiostatinantibodies.

It is an object of present invention to provide a method for detectingand quantifying the presence of an antibody specific for an angiostatinin a body fluid.

Still another object of the present invention is to provide acomposition consisting of antibodies to angiostatin that are selectivefor specific regions of the angiostatin molecule that do not recognizeplasminogen.

It is another object of the present invention to provide a method forthe detection or prognosis of cancer.

It is another object of the present invention to provide a compositionfor use in visualizing and quantitating sites of angiostatin binding invivo and in vitro.

It is yet another object of the present invention to provide acomposition for use in detection and quantification of angiostatinbiosynthesis.

It is yet another object of the present invention to provide a therapyfor cancer that has minimal side effects.

Still another object of the present invention is to provide acomposition comprising angiostatin or an angiostatin protein linked to acytotoxic agent for treating or repressing the growth of a cancer.

Another object of the present invention is to provide a method fortargeted delivery of angiostatin-related compositions to specificlocations.

Yet another object of the invention is to provide compositions andmethods useful for gene therapy for the modulation of angiogenicprocesses.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows SEQ ID NO:1, the amino acid sequence of the whole murineplasminogen.

FIG. 2 shows the beginning sequence of the angiostatin for murine (SEQID NO:2) and compares the murine sequence with corresponding human (SEQID NO:3), Rhesus monkey (SEQ ID NO:4), porcine (SEQ ID NO:5) and bovine(SEQ ID NO:6) plasminogen protein fragments. The mouse sequence islisted first, followed by human, Rhesus, porcine and bovine.

FIG. 3 shows BrdU labeling index of tumor cells in the lung in thepresence or absence of a primary tumor.

FIG. 4 shows Matrigel analysis of the influence of a Lewis lung primarytumor on bFGF driven angiogenesis in vivo.

FIG. 5 shows dose response curve for serum derived from mice bearingLewis lung carcinoma (LLC-Low) versus serum from normal mice. Bovinecapillary endothelial cells were assayed in a bFGF-driven 72-hourproliferation assay.

FIG. 6 shows that both low and high metastatic tumors containendothelial mitogenic activity in their ascites, but only the lowmetastatic tumor line has endothelial inhibitory activity in the serum.

FIG. 7 shows a C4 Reverse Phase Chromatographic profile of partiallypurified serum or urine from tumor-bearing animals.

FIG. 8 shows surface lung metastases after the 13 day treatment of micewith intact plasminogen molecule, active fraction from a lysine bindingsite I preparation of human plasminogen, concentrated urine from tumorbearing mice and concentrated urine from normal mice.

FIG. 9 shows lung weight after the 13 day treatment of mice with intactplasminogen molecule of human plasminogen, active fraction from lysinebinding site I preparation, concentrated urine from tumor bearing miceand concentrated urine from normal mice.

FIG. 10 is a schematic representation of the pTrcHis vector.

FIG. 11 depicts an immunoblot of E. coli expressed human angiostatinfrom a 10 L scaled-up fermentation, probed with monoclonal antibodyagainst human plasminogen kringle region 1-3. Arrow shows recombinanthuman angiostatin. A) shows recombinant angiostatin eluted with 0.2 Mamino caproic acid; B) shows the last wash with 1×PBS of the lysinecolumn; and C) shows clarified lysate from cracked cells.

FIG. 12. Is a graph depicting percent inhibition of growing bovinecapillary endothelial cells as a function of dilution of stock; A1, A2,B1, B2, and E are recombinant clones that express human angiostatinanit-angiogenesis activity; C1, C2, D1 and D2 controls are negativecontrols clones containing vector only without the human DNA sequencecoding for angiostatin.

FIG. 13 shows the inhibitory effect on proliferation of recombinanthuman angiostatin on bovine capillary endothelial cells in vitro.

FIG. 14 shows the growth proliferation index and apoptotic index afterremoval of the primary tumor and treatment with saline or a fumagillinanalogue with anti-angiogenic activity

FIG. 15 shows the inhibition of growth of a T241 primary tumor in miceby treatment with human angiostatin in vivo with a single injection of40 mg/kg/day.

FIG. 16 shows the inhibition of growth of a LLC-LM primary tumor in miceby treatment with human angiostatin in vivo at two doses of 40 mg/kg perdose (80 mg/kg/day).

FIG. 17 shows the effect of the removal of a Lewis lung carcinomaprimary tumor on the growth of its lung metastases.

FIG. 18 shows the growth proliferation and apoptotic index after tumorresection

FIG. 19 shows the effect of administration of angiostatin protein tomice having implated T241 fibrosarcoma cells on total tumor volume as afunction of time.

FIG. 20 shows the effect of administration of angiostatin protein tomice having implated Lewis lung carcinoma (LM) cells on total tumorvolume as a function of time.

FIG. 21 shows the effect of administration of angiostatin protein tomice having implated reticulum cell sarcoma cells on total tumor volumeas a function of time.

FIG. 22 shows the effect of administration of angiostatin protein toimmunodeficient SCID mice having implated human prostate carcinoma PC-3cells on total tumor volume as a function of time over a 24 day period.

FIG. 23 shows the effect of administration of angiostatin protein toimmunodeficient SCID mice having implated human breast carcinoma MDA-MBcells on total tumor volume as a function of time over a 24 day period.

FIG. 24 is a schematic representation of cloning of the mouse DNAsequence coding for mouse angiostatin protein derived from mouseplasminogen cDNA. The mouse angiostatin encompasses mouse plasminogenkringle regions 1-4. PCR means polymerase chain reaction; P1 is the5′-end oligonucleotide primer for PCR; P2 is the 3′-end oligonucleotideprimer for PCR; SS designates the signal sequence; ATG is thetranslation initiation codon; TAA is the translation stop codon; HArepresents the hemagglutinin epitope tag (YPYDVPDYASL); K1, K2, K3 andK4 represent mouse plasminogen kringle regions 1, 2, 3 and 4respectively. CMV is the cytomegalovirus promoter; T7 is the bacteriaphage promoter; PA represents pre-activation proteins; and SP6 is the Sp6 promoter.

FIG. 25 depicts the number of cells as a function of days fornon-transfected cells (mock); cells transfected with the vector alone,without the DNA sequence coding for angiostatin (Vector 5), and twoangiostatin expressing clones (AST 31 and AST 37). Panel (a) representsthe results of transfection of T241 cells. Panel (b) represents theresults of LL2 cells.

FIG. 26 shows the results of culture medium derived from E. coli cellscontaining the angiostatin clone on cell number. Non-transfected cells(mock); cells transfected with the vector alone, without the DNAsequence coding for angiostatin (Vector 5), and three angiostatinexpressing clones (AST 25, AST 31 and AST 37). Panel (a) represents theresults of incubation of culture medium from control (mock) and allangiostatin clones (expressing and non-expressing) on cell number. Panel(b) represents the results of incubation of culture medium from control(mock), vector alone (vector 6) and angiostatin clones expressing mouseangiostatin on cell number. Panel (c) represents the results ofincubation of purified culture medium from control (mock) andangiostatin clones expressing mouse angiostatin on cell number, whereinthe culture medium was purified over a lysine-sepharose colume to yieldlysine binding components.

FIG. 27 shows the effect on total tumor volume as a function of time ofimplanting T241 fibrosarcoma cells in mice, where the fibrosarcoma cellshave been transfected with a vector containing a DNA sequence coding forangiostatin protein, and where the vector is capable of expressingangiostatin protein. “Non-transfected” represents unaltered T241fibrosarcoma cells implanted in mice. “Vector 6” represents T241fibrosarcoma cells transfected with the vector only, which does notcontain the DNA sequence coding for angiostatin protein, implanted inmice. “Clone 25, Clone 31 and Clone 37” represent threeangiostatin-producing clones of T241 fibrosarcoma cells transfected witha vector containing the DNA sequence coding for angiostation proteinimplanted in mice.

FIG. 28 shows a schematic representation of the structure of humanplasminogen and its kringle fragments. Human plaminogen is a singlechain protein containing 791 amino acids with one side of N-linkedglycosylation at Asn²⁸⁹. The non-protease region of human plasminogenconsisting of the N-terminal 561 amino acids existing in five separatedomains, termed kringles as shown in circles (K1, K2, K3, K4 and K5),along with proteins that separate these structures. Each tripledisulfide bonded kringle contains 80 amino acids. Angiostatin covers thefirst 4 of these kringle domains (K1-4), kringle 3 (K1-3) and kringle 4(K4) are obtained by digestion of human plasminogen with elastase. Therest of the kringle fragments are recombinant proteins expressed in E.coli. SS=signal sequence. PA=preactivation protein.

FIG. 29 shows a SDS-PAGE analysis of purified recombinant and nativekringle fragments of plasminogen under reducing conditions. (A)Individual recombinant kringle fragments purified from E. coli bacteriallysates were loaded onto a 15% SDS gel followed by staining withCoomassie blue. Approximately 5 μg of each protein was loaded per lane.(lane 2=kringle 1 (K1); lane 3=kringle 2 (K2); lane 4=kringle 3 (K3);lane 5=kringle 4 (K4); lane 1=molecular weight markers). (B) Purifiedlarge kringle fragments were stained with Coomassie blue. Kringles 1-4(lane 2) and kringles 1-3 (lane 3) were obtained by digestion of humanplasminogen with elastase and purified by lysine-Sepharosechromatography. Recombinant fragment of kringles 2-3 (lane 4) wasexpressed in E. coli and re-folded in vitro. Molecular weight markersare indicated on the left (lane 1).

FIG. 30 shows an inhibition of endothelial cell proliferation byrecombinant individual kringle fragments of angiostatin. Kringlefragments were assayed on bovine capillary endothelial cells in thepresence of 1 ng/ml bFGF for 72 hours. (A) Anti-endothelial cellproliferative effects of two lysine-binding kringles, rK1 and rK4. Thehigh-affinity lysine binding kringle, K1 (-o-), inhibited BCE cellproliferation in a dose-dependent manner. The intermediate-affinitylysine binding kringle, K4 (--), showed only little inhibitory effectat high concentrations. (B) Inhibition of BCE cell proliferation bynon-lysine binding K2 and K3. Both K2 (-▪-) and K3 (

) inhibited BCE cell proliferation in a dose-dependent manner. Datarepresents the mean+/−SEM of triplicates.

FIG. 31 shows an anti-endothelial proliferation activity of largekringle fragments of angiostatin. Proteolytic fragments, K1-4(angiostatin) (-o-) and K1-3 (-▪-), inhibited BCE cell proliferation ina dose-dependent manner. Recombinant K2-3 (--) fragments exhibited aless potent inhibition than those of K1-3 and K1-4. Data represents themean of three determinations (+/−SEM) as percentages of inhibition.

FIG. 32 shows an additive inhibitory activity of recombinant kringle 2and kringle 3. (A) The intact fragment of rK2-3 (also see FIG. 31)displayed a weak inhibitory effect only at the concentration of 320 nM.At the same concentration, an additive inhibition was seen when mutantfragments of rK2 cysteine replaced by serine at the position of 169) andK3 (cysteine replaced by serine at the position of 297) were assayedtogether on BCE cells. Each value represents the mean+/−SEM oftriplicates. (B) Schematic structure and amino acid sequence of K2 andK3. An inter-chain kringle disulfide bond was previously reported to bepresent between cysteine¹⁶⁹ of K2 and cysteine²⁹⁷ of K3 (Söhndel, S.,Hu, C.-K., Marti, D., Affolter, M., Schaller, J., Llinas, M., andRickli, E. E. (1996) Biochem. in press).

FIG. 33 shows an inhibition of endothelial proliferation bycombinatorial kringle fragments. The assay was performed with aconcentration of 320 nM for each kringle fragment. Values represent themean of three determinations (+/−SEM) as percentages of inhibition. (A)Inhibitory effects of fragments by combination of various individualkringles. (B) Combinatorial inhibitory activity of combined kringlefragments.

FIG. 34 shows an inhibitory activity of angiostatin on endothelial cellsafter reduction and alkylation. (A) SDS-PAGE analysis of the reduced(lane 2) and non-reduced (lane 1) forms of human angiostatin. Purifiedhuman angiostatin was reduced with DTT followed by alkylation of theprotein with an excess amount of iodoacetamide. The treated samples weredialyzed and assayed on BCE cells. (B) Inhibition of BCE cellproliferation by reduced and non-reduced forms of angiostatin at aconcentration of 320 nM. Data represents the mean of inhibition+/−SEM oftriplicates.

FIG. 35 shows an amino acid sequence alignment of putative kringledomains of human angiostatin. The sequences of four kringle domains werealigned according to their conserved cysteines. Identical and conservedamino acids are shaded. The boxed amino acids in kringle 4 show thepositively charged double lysines adjacent to conserved cysteineresidues of 22 and 80.

FIG. 36 shows lysine-binding characteristics and reactivity of expressedangiostatin.

FIG. 36A shows a Coomassie stained gel (40 μl load).

FIG. 36B shows an immunoblot (20 μl load) of similar gel. Lane: 1 showsbroth from shake flasks of induced cultures showing angiostatin proteinat about 50 kD and a few other proteins. Broth from induced cultures isdiluted 1:1 with buffer and directly loaded onto lysine-sepharose. Lane:2 shows the unbound fraction that passed through the lysine column. Allangiostatin protein expressed by P. pastoris binds to the lysine column.Lane: 3 shows specific elution with 0.2 M amino caproic acid showingthat P. pastoris expressed angiostatin protein binds lysine and can bepurified in a single step to homogeneity over a lysine-sepharose. Also,the P. pastoris expressed angiostatin protein is recognized by aconformationally dependent monoclonal antibody (VAP) raised againstkringles 1 to 3.

FIG. 37 shows P. pastoris expressed angiostatin protein is seen as adoublet that migrates at 49 kD and 51.5 kD on denatured unreducedSDS-PAGE Coomassie stained gels. Removing the single N-linked complexchain from the expressed angiostatin protein with N-glycanase specificfor high mannose structures results in a single band of 49.5 kD. Panel Aand panel B show a Coomassie stained gel and an immunoblot of a similargel respectively. Lane: 1 shows a purified P. pastoris expressedangiostatin protein. Lane: 2 shows a purified P. pastoris expressedangiostatin protein incubated in digestion conditions withoutN-glycanase. Lane: 3 shows purified P. pastoris expressed angiostatinprotein digested with N-glycanase.

FIG. 38A shows 4 μg of purified P. pastoris expressed angiostatinprotein as a doublet on a Coomassie gel.

FIG. 38B shows that the purified recombinant inhibits BCE proliferation.The BCE assay cell counts obtained after 72 hours is shown, in thepresence () or absence (o) of bFGF, and in the presence of bFGF withPBS as control (Δ), and in the presence of bFGF with P. pastorisexpressed angiostatin protein (▴).

FIG. 38C shows that the inhibition is dose dependent.

FIG. 39 shows P. pastoris expressed purified angiostatin was givensystemically (subcutaneous) to mice with primary tumors.

FIGS. 39A and B show the number of metastases and the lung weightsrespectively of mice treated daily with saline or P. pastoris expressedangiostatin or plasminogen derived angiostatin protein. In contrast tothe lungs of mice treated with saline, lungs of mice treated with P.pastoris expressed angiostatin protein or with plasminogen derivedangiostatin protein were non-vascularized and metastases were potentlysuppressed.

FIG. 40 shows that the lungs of mice treated with P. pastoris expressedangiostatin were pink with micrometastases while the lungs of the salinecontrol group were completely covered with vascularized metastases.

DETAILED DESCRIPTION

The present invention includes compositions and methods for thedetection and treatment of diseases and processes that are mediated byor associated with angiogenesis. The composition is angiostatin, whichcan be isolated from body fluids including, but not limited to, serum,urine and ascites, or synthesized by chemical or biological methods(e.g. cell culture, recombinant gene expression, protein synthesis, andin vitro enzymatic catalysis of plasminogen or plasmin to yield activeangiostatin). Recombinant techniques include gene amplification from DNAsources using the polymerase chain reaction (PCR), and geneamplification from RNA sources using reverse transcriptase/PCR.Angiostatin inhibits the growth of blood vessels into tissues such asunvascularized or vascularized tumors.

The present invention also encompasses a composition comprising, avector containing a DNA sequence encoding angiostatin, wherein thevector is capable of expressing angiostatin when present in a cell, acomposition comprising a cell containing a vector, wherein the vectorcontains a DNA sequence encoding angiostatin or fragments or analogsthereof, and wherein the vector is capable of expressing angiostatinwhen present in the cell, and a method comprising, implanting into ahuman or non-human animal a cell containing a vector, wherein the vectorcontains a DNA sequence encoding angiostatin, and wherein the vector iscapable of expressing angiostatin when present in the cell.

Still further, the present invention encompasses angiostatin,angiostatin fragments, angiostatin antisera, angiostatin receptoragonists or angiostatin receptor antagonists that are combined withpharmaceutically acceptable excipients, and optionally sustained-releasecompounds or compositions, such as biodegradable polymers, to formtherapeutic compositions. In particular, the invention includes acomposition comprising an antibody that specifically binds toangiostatin, wherein the antibody does not bind to plasminogen.

More particularly, the present invention includes a protein designatedangiostatin that has a molecular weight of approximately 38 to 45kilodaltons (kD) that is capable of overcoming the angiogenic activityof endogenous growth factors such as bFGF, in vitro. Angiostatin is aprotein having a molecular weight of between approximately 38kilodaltons and 45 kilodaltons as determined by reducing polyacrylamidegel electrophoresis and having an amino acid sequence substantiallysimilar to that of a murine plasminogen fragment beginning at amino acidnumber 98 of an intact murine plasminogen molecule. The term“substantially similar,” when used in reference to angiostatin aminoacid sequences, means an amino acid sequence having anti-angiogenicactivity and having a molecular weight of approximately 38 kD to 45 kD,which also has a high degree of sequence homology to the proteinfragment of mouse plasminogen beginning approximately at amino acidnumber 98 in mouse plasminogen and weighing 38 kD to 45 kD. A highdegree of homology means at least approximately 60% amino acid homology,desirably at least approximately 70% amino acid homology, and moredesirably at least approximately 80% amino acid homology. The term“endothelial inhibiting activity” as used herein means the capability ofa molecule to inhibit angiogenesis in general and, for example, toinhibit the growth of bovine capillary endothelial cells in culture inthe presence of fibroblast growth factor.

The amino acid sequence of the complete murine plasminogen molecule isshown in FIG. 1 and in SEQ ID NO:1, The sequence for angiostatin beginsapproximately at amino acid 98. Active human angiostatin may start ateither amino acid 97 or 99 of the intact human plasminogen molecule. Theamino acid sequence of the first 339 amino acids of angiostatin frommouse is shown in FIG. 2, (SEQ ID NO:2), and is compared with thesequences of corresponding plasminogen protein fragments from human (SEQID NO:3, Rhesus monkey (SEQ ID NO:4), porcine (SEQ ID NO:5) and bovine(SEQ ID NO:6) plasminogen. Given that these sequences are identical inwell over 50% of their amino acids, it is to be understood that theamino acid sequence of the angiostatin is substantially similar amongspecies. The total number of amino acids in angiostatin is not knownprecisely but is defined by the molecular weight of the active molecule.The amino acid sequence of the angiostatin of the present invention mayvary depending upon from which species the plasminogen molecule isderived. Thus, although the angiostatin of the present invention that isderived from human plasminogen has a slightly different sequence thanangiostatin derived from mouse, it has anti-angiogenic activity as shownin a mouse tumor model.

Angiostatin has been shown to be capable of inhibiting the growth ofendothelial cells in vitro. Angiostatin does not inhibit the growth ofcell lines derived from other cell types. Specifically, angiostatin hasno effect on Lewis lung carcinoma cell lines, mink lung epithelium, 3T3fibroblasts, bovine aortic smooth muscle cells, bovine retinal pigmentepithelium, MDCk cells (canine renal epithelium), WI38 cells (humanfetal lung fibroblasts) EFN cells (murine fetal fibroblasts) and LMcells (murine connective tissue). Endogenous angiostatin in a tumorbering mouse is effective at inhibiting metastases at a systemicconcentration of approximately 10 mg angiostatin/kg body weight.

Angiostatin has a specific three dimensional conformation that isdefined by the kringle region of the plasminogen molecule. (Robbins, K.C., “The plasminogen-plasmin enzyme system” Hemostasis and Thrombosis,Basic Principles and Practice, 2nd Edition, ed. by Colman, R. W. et al.J.B. Lippincott Company, pp. 340-357, 1987) There are five such kringleregions, which are conformationally related motifs and have substantialsequence homology, in the NH₂ terminal portion of the plasminogenmolecule. The three dimensional conformation of angiostatin is believedto encompass plasminogen kringle regions 1 through 3 and a part ofkringle region 4. Each kringle region of the plasminogen moleculecontains approximately 80 amino acids and contains 3 disulfide bonds.This cysteine motif is known to exist in other biologically activeproteins. These proteins include, but are not limited to, prothrombin,hepatocyte growth factor, scatter factor and macrophage stimulatingprotein. (Yoshimura, T, et al., “Cloning, sequencing, and expression ofhuman macrophage stimulating protein (MSP, MST1) confirms MSP as amember of the family of kringle proteins and locates the MSP gene onChromosome 3” J. Biol. Chem., Vol. 268, No. 21, pp. 15461-15468, 1993).It is contemplated that any isolated protein or protein having a threedimensional kringle-like conformation or cysteine motif that hasanti-angiogenic activity in vivo, is part of the present invention.

The present invention also includes the detection of the angiostatin inbody fluids and tissues for the purpose of diagnosis or prognosis ofdiseases such as cancer. The present invention also includes thedetection of angiostatin binding sites and receptors in cells andtissues. The present invention also includes methods of treating orpreventing angiogenic diseases and processes including, but not limitedto, arthritis and tumors by stimulating the production of angiostatin,and/or by administering substantially purified angiostatin, orangiostatin agonists or antagonists, and/or angiostatin antisera orantisera directed against angiostatin antisera to a patient. Additionaltreatment methods include administration of angiostatin, angiostatinfragments, angiostatin analogs, angiostatin antisera, or angiostatinreceptor agonists and antagonists linked to cytotoxic agents. It is tobe understood that the angiostatin can be animal or human in origin.Angiostatin can also be produced synthetically by chemical reaction orby recombinant techniques in conjunction with expression systems.Angiostatin can also be produced by enzymatically cleaving isolatedplasminogen or plasmin to generate proteins having anti-angiogenicactivity. Angiostatin may also be produced by compounds that mimic theaction of endogenous enzymes that cleave plasminogen to angiostatin.Angiostatin production may also be modulated by compounds that affectthe activity of plasminogen cleaving enzymes.

Passive antibody therapy using antibodies that specifically bindangiostatin can be employed to modulate angiogenic-dependent processessuch as reproduction, development, and wound healing and tissue repair.In addition, antisera directed to the Fab regions of angiostatinantibodies can be administered to block the ability of endogenousangiostatin antisera to bind angiostatin.

The present invention also encompasses gene therapy whereby the geneencoding angiostatin is regulated in a patient. Various methods oftransferring or delivering DNA to cells for expression of the geneproduct protein, otherwise referred to as gene therapy, are disclosed inGene Transfer into Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev.Biotechn. 12(4): 335-356 (1992), which is hereby incorporated byreference. Gene therapy encompasses incorporation of DNA sequences intosomatic cells or germ line cells for use in either ex vivo or in vivotherapy. Gene therapy functions to replace genes, augment normal orabnormal gene function, and to combat infectious diseases and otherpathologies.

Strategies for treating these medical problems with gene therapy includetherapeutic strategies such as identifying the defective gene and thenadding a functional gene to either replace the function of the defectivegene or to augment a slightly functional gene; or prophylacticstrategies, such as adding a gene for the product protein that willtreat the condition or that will make the tissue or organ moresusceptible to a treatment regimen. As an example of a prophylacticstrategy, a gene such as angiostatin may be placed in a patient and thusprevent occurrence of angiogenesis; or a gene that makes tumor cellsmore susceptible to radiation could be inserted and then radiation ofthe tumor would cause increased killing of the tumor cells.

Many protocols for transfer of angiostatin DNA or angiostatin regulatorysequences are envisioned in this invention. Transfection of promotersequences, other than one normally found specifically associated withangiostatin, or other sequences which would increase production ofangiostatin protein are also envisioned as methods of gene therapy. Anexample of this technology is found in Transkaryotic Therapies, Inc., ofCambridge, Mass., using homologous recombination to insert a “geneticswitch” that turns on an erythropoietin gene in cells. See GeneticEngineering News, Apr. 15, 1994. Such “genetic switches” could be usedto activate angiostatin (or the angiostatin receptor) in cells notnormally expressing angiostatin (or the angiostatin receptor).

Gene transfer methods for gene therapy fall into three broadcategories-physical (e.g., electroporation, direct gene transfer andparticle bombardment), chemical (lipid-based carriers, or othernon-viral vectors) and biological (virus-derived vector and receptoruptake). For example, non-viral vectors may be used which includeliposomes coated with DNA. Such liposome/DNA complexes may be directlyinjected intravenously into the patient. It is believed that theliposome/DNA complexes are concentrated in the liver where they deliverthe DNA to macrophages and Kupffer cells. These cells are long lived andthus provide long term expression of the delivered DNA. Additionally,vectors or the “naked” DNA of the gene may be directly injected into thedesired organ, tissue or tumor for targeted delivery of the therapeuticDNA.

Gene therapy methodologies can also be described by delivery site.Fundamental ways to deliver genes include ex vivo gene transfer, in vivogene transfer, and in vitro gene transfer. In ex vivo gene transfer,cells are taken from the patient and grown in cell culture. The DNA istransfected into the cells, the transfected cells are expanded in numberand then reimplanted in the patient. In in vitro gene transfer, thetransformed cells are cells growing in culture, such as tissue culturecells, and not particular cells from a particular patient. These“laboratory cells” are transfected, the transfected cells are selectedand expanded for either implantation into a patient or for other uses.

In vivo gene transfer involves introducing the DNA into the cells of thepatient when the cells are within the patient. Methods include usingvirally mediated gene transfer using a noninfectious virus to deliverthe gene in the patient or injecting naked DNA into a site in thepatient and the DNA is taken up by a percentage of cells in which thegene product protein is expressed. Additionally, the other methodsdescribed herein, such as use of a “gene gun,” may be used for in vitroinsertion of angiostatin DNA or angiostatin regulatory sequences.

Chemical methods of gene therapy may involve a lipid based compound, notnecessarily a liposome, to ferry the DNA across the cell membrane.Lipofectins or cytofectins, lipid-based positive ions that bind tonegatively charged DNA, make a complex that can cross the cell membraneand provide the DNA into the interior of the cell. Another chemicalmethod uses receptor-based endocytosis, which involves binding aspecific ligand to a cell surface receptor and enveloping andtransporting it across the cell membrane. The ligand binds to the DNAand the whole complex is transported into the cell. The ligand genecomplex is injected into the blood stream and then target cells thathave the receptor will specifically bind the ligand and transport theligand-DNA complex into the cell.

Many gene therapy methodologies employ viral vectors to insert genesinto cells. For example, altered retrovirus vectors have been used in exvivo methods to introduce genes into peripheral and tumor-infiltratinglymphocytes, hepatocytes, epidermal cells, myocytes, or other somaticcells. These altered cells are then introduced into the patient toprovide the gene product from the inserted DNA.

Viral vectors have also been used to insert genes into cells using invivo protocols. To direct tissue-specific expression of foreign genes,cis-acting regulatory elements or promoters that are known to be tissuespecific can be used. Alternatively, this can be achieved using in situdelivery of DNA or viral vectors to specific anatomical sites in vivo.For example, gene transfer to blood vessels in vivo was achieved byimplanting in vitro transduced endothelial cells in chosen sites onarterial walls. The virus infected surrounding cells which alsoexpressed the gene product. A viral vector can be delivered directly tothe in vivo site, by a catheter for example, thus allowing only certainareas to be infected by the virus, and providing long-term, sitespecific gene expression. In vivo gene transfer using retrovirus vectorshas also been demonstrated in mammary tissue and hepatic tissue byinjection of the altered virus into blood vessels leading to the organs.

Viral vectors that have been used for gene therapy protocols include butare not limited to, retroviruses, other RNA viruses such as poliovirusor Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV40, vaccinia and other DNA viruses. Replication-defective murineretroviral vectors are the most widely utilized gene transfer vectors.Murine leukemia retroviruses are composed of a single strand RNAcomplexed with a nuclear core protein and polymerase (pol) enzymes,encased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include the gag, pol, and env genes enclosed at by the 5′and 3′ long terminal repeats (LTR). Retroviral vector systems exploitthe fact that a minimal vector containing the 5′ and 3′ LTRs and thepackaging signal are sufficient to allow vector packaging, infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line. Fundamentaladvantages of retroviral vectors for gene transfer include efficientinfection and gene expression in most cell types, precise single copyvector integration into target cell chromosomal DNA, and ease ofmanipulation of the retroviral genome.

The adenovirus is composed of linear, double stranded DNA complexed withcore proteins and surrounded with capsid proteins. Advances in molecularvirology have led to the ability to exploit the biology of theseorganisms to create vectors capable of transducing novel geneticsequences into target cells in vivo. Adenoviral-based vectors willexpress gene product proteins at high levels. Adenoviral vectors havehigh efficiencies of infectivity, even with low titers of virus.Additionally, the virus is fully infective as a cell free virion soinjection of producer cell lines are not necessary. Another potentialadvantage to adenoviral vectors is the ability to achieve long termexpression of heterologous genes in vivo.

Mechanical methods of DNA delivery include fusogenic lipid vesicles suchas liposomes or other vesicles for membrane fusion, lipid particles ofDNA incorporating cationic lipid such as lipofectin, polylysine-mediatedtransfer of DNA, direct injection of DNA, such as microinjection of DNAinto germ or somatic cells, pneumatically delivered DNA-coatedparticles, such as the gold particles used in a “gene gun,” andinorganic chemical approaches such as calcium phosphate transfection.Another method, ligand-mediated gene therapy, involves complexing theDNA with specific ligands to form ligand-DNA conjugates, to direct theDNA to a specific cell or tissue.

It has been found that injecting plasmid DNA into muscle cells yieldshigh percentage of the cells which are transfected and have sustainedexpression of marker genes. The DNA of the plasmid may or may notintegrate into the genome of the cells. Non-integration of thetransfected DNA would allow the transfection and expression of geneproduct proteins in terminally differentiated, non-proliferative tissuesfor a prolonged period of time without fear of mutational insertions,deletions, or alterations in the cellular or mitochondrial genome.Long-term, but not necessarily permanent, transfer of therapeutic genesinto specific cells may provide treatments for genetic diseases or forprophylactic use. The DNA could be reinjected periodically to maintainthe gene product level without mutations occurring in the genomes of therecipient cells. Non-integration of exogenous DNAs may allow for thepresence of several different exogenous DNA constructs within one cellwith all of the constructs expressing various gene products.

Particle-mediated gene transfer methods were first used in transformingplant tissue. With a particle bombardment device, or “gene gun,” amotive force is generated to accelerate DNA-coated high densityparticles (such as gold or tungsten) to a high velocity that allowspenetration of the target organs, tissues or cells. Particle bombardmentcan be used in in vitro systems, or with ex vivo or in vivo techniquesto introduce DNA into cells, tissues or organs.

Electroporation for gene transfer uses an electrical current to makecells or tissues susceptible to electroporation-mediated gene transfer.A brief electric impulse with a given field strength is used to increasethe permeability of a membrane in such a way that DNA molecules canpenetrate into the cells. This technique can be used in in vitrosystems, or with ex vivo or in vivo techniques to introduce DNA intocells, tissues or organs.

Carrier mediated gene transfer in vivo can be used to transfect foreignDNA into cells. The carrier-DNA complex can be conveniently introducedinto body fluids or the bloodstream and then site specifically directedto the target organ or tissue in the body. Both liposomes andpolycations, such as polylysine, lipofectins or cytofectins, can beused. Liposomes can be developed which are cell specific or organspecific and thus the foreign DNA carried by the liposome will be takenup by target cells. Injection of immunoliposomes that are targeted to aspecific receptor on certain cells can be used as a convenient method ofinserting the DNA into the cells bearing the receptor. Another carriersystem that has been used is the asialoglycoprotein/polylysine conjugatesystem for carrying DNA to hepatocytes for in vivo gene transfer.

The transfected DNA may also be complexed with other kinds of carriersso that the DNA is carried to the recipient cell and then resides in thecytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclearproteins in specifically engineered vesicle complexes and carrieddirectly into the nucleus.

Gene regulation of angiostatin may be accomplished by administeringcompounds that bind to the angiostatin gene, or control regionsassociated with the angiostatin gene, or its corresponding RNAtranscript to modify the rate of transcription or translation.Additionally, cells transfected with a DNA sequence encoding angiostatinmay be administered to a patient to provide an in vivo source ofangiostatin. For example, cells may be transfected with a vectorcontaining a nucleic acid sequence encoding angiostatin.

The term “vector” as used herein means a carrier that can contain orassociate with specific nucleic acid sequences, which functions totransport the specific nucleic acid sequences into a cell. Examples ofvectors include plasmids and infective microorganisms such as viruses,or non-viral vectors such as ligand-DNA conjugates, liposomes, lipid-DNAcomplexes. It may be desirable that a recombinant DNA moleculecomprising an angiostatin DNA sequence is operatively linked to anexpression control sequence to form an expression vector capable ofexpressing angiostatin. The transfected cells may be cells derived fromthe patient's normal tissue, the patient's diseased tissue, or may benon-patient cells.

For example, tumor cells removed from a patient can be transfected witha vector capable of expressing the angiostatin protein of the presentinvention, and re-introduced into the patient. The transfected tumorcells produce angiostatin levels in the patient that inhibit the growthof the tumor. Patients may be human or non-human animals. Cells may alsobe transfected by non-vector, or physical or chemical methods known inthe art such as electroporation, ionoporation, or via a “gene gun.”Additionally, angiostatin DNA may be directly injected, without the aidof a carrier, into a patient. In particular, angiostatin DNA may beinjected into skin, muscle or blood.

The gene therapy protocol for transfecting angiostatin into a patientmay either be through integration of the angiostatin DNA into the genomeof the cells, into minichromosomes or as a separate replicating ornon-replicating DNA construct in the cytoplasm or nucleoplasm of thecell. Angiostatin expression may continue for a long-period of time ormay be reinjected periodically to maintain a desired level of theangiostatin protein in the cell, the tissue or organ or a determinedblood level.

Angiostatin can be isolated on an HPLC C4 column (see Table 3). Theangiostatin protein is eluted at 30 to 35% in an acetonitrile gradient.On a sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE)gel under reducing conditions, the protein band with activity eluted asa single peak at approximately 38 kilodaltons.

The inventors have shown that a growing primary tumor is associated withthe release into the blood stream of specific inhibitor(s) ofendothelial cell proliferation, including angiostatin which can suppressangiogenesis within a metastasis and thereby inhibit the growth of themetastasis itself. The source of the angiostatin associated with theprimary tumor is not known. The compound may be produced by degradationof plasminogen by a specific protease, or angiostatin could be producedby expression of a specific gene coding for angiostatin.

The angiogenic phenotype of a primary tumor depends on production ofangiogenic proteins in excess of endothelial cell inhibitors which areelaborated by normal cells, but are believed to be down-regulated duringtransformation to neoplasia. While production of angiostatin may bedown-regulated in an individual tumor cell relative to production by itsparent cell type, the total amount of inhibitor elaborated by the wholetumor may be sufficient to enter the circulation and suppressendothelial growth at remote sites of micrometastases. Angiostatinremains in the circulation for a significantly longer time than theangiogenic protein(s) released by a primary tumor. Thus, the angiogenicproteins appear to act locally, whereas angiostatin acts globally andcirculates in the blood with a relatively long half-life. The half-lifeof the angiostatin is approximately 12 hours to 5 days.

Although not wanting to be bound by the following hypothesis, it isbelieved that when a tumor becomes angiogenic it releases one or moreangiogenic proteins (e.g., aFGF, bFGF, VEGF, IL-8, GM-CSF, etc.), whichact locally, target endothelium in the neighborhood of a primary tumorfrom an extravascular direction, and do not circulate (or circulate witha short half-life). These angiogenic proteins must be produced in anamount sufficient to overcome the action of endothelial cell inhibitor(inhibitors of angiogenesis) for a primary tumor to continue to expandits population. Once such a primary tumor is growing well, it continuesto release endothelial cell inhibitors into the circulation. Accordingto this hypothesis, these inhibitors act remotely at a distance from theprimary tumor, target capillary endothelium of a metastasis from anintravascular direction, and continue to circulate. Thus, just at thetime when a remote metastasis might begin to initiate angiogenesis, thecapillary endothelium in its neighborhood could be inhibited by incomingangiostatin.

Once a primary tumor has reached sufficient size to cause angiostatin tobe released continuously into the circulation, it is difficult for asecond tumor implant (or a micrometastasis) to initiate or increase itsown angiogenesis. If a second tumor implant (e.g., into the subcutaneousspace, or into the cornea, or intravenously to the lung) occurs shortlyafter the primary tumor is implanted, the primary tumor will not be ableto suppress the secondary tumor (because angiogenesis in the secondarytumor will already be well underway). If two tumors are implantedsimultaneously (e.g., in opposite flanks), the inhibitors may have anequivalent inhibiting effect on each other.

The angiostatin of the present invention can be:

(i) Administered to tumor-bearing humans or animals as anti-angiogenictherapy;

(ii) Monitored in human or animal serum, urine, or tissues as prognosticmarkers; and

(iii) Used as the basis to analyze serum and urine of cancer patientsfor similar angiostatic molecules.

It is contemplated as part of the present invention that angiostatin canbe isolated from a body fluid such as blood or urine of patients or theangiostatin can be produced by recombinant DNA methods or syntheticprotein chemical methods that are well known to those of ordinary skillin the art. Protein purification methods are well known in the art and aspecific example of a method for purifying angiostatin, and assaying forinhibitor activity is provided in the examples below. Isolation of humanendogenous angiostatin is accomplished using similar techniques.

One example of a method of producing angiostatin using recombinant DNAtechniques entails the steps of (1) identifying and purifyingangiostatin as discussed above, and as more fully described below, (2)determining the N-terminal amino acid sequence of the purifiedinhibitor, (3) synthetically generating 5′ and 3′ DNA oligonucleotideprimers for the angiostatin sequence, (4) amplifying the angiostatingene sequence using polymerase, (5) inserting the amplified sequenceinto an appropriate vector such as an expression vector, (6) insertingthe gene containing vector into a microorganism or other expressionsystem capable of expressing the inhibitor gene, and (7) isolating therecombinantly produced inhibitor. Appropriate vectors include viral,bacterial and eukaryotic (such as yeast) expression vectors. The abovetechniques are more fully described in laboratory manuals such as“Molecular Cloning: A Laboratory Manual” Second Edition by Sambrook etal., Cold Spring Harbor Press, 1989. The DNA sequence of humanplasminogen has been published (Browne, M. J., et al., “Expression ofrecombinant human plasminogen and aglycoplasminogen in HeLa cells”Fibrinolysis Vol. 5 (4). 257-260, 1991) and is incorporated herein byreference

The gene for angiostatin may also be isolated from cells or tissue (suchas tumor cells) that express high levels of angiostatin by (1) isolatingmessenger RNA from the tissue, (2) using reverse transcriptase togenerate the corresponding DNA sequence and then (3) using thepolymerase chain reaction (PCR) with the appropriate primers to amplifythe DNA sequence coding for the active angiostatin amino acid sequence.

Yet another method of producing angiostatin, or biologically activefragments thereof, is by protein synthesis. Once a biologically activefragment of an angiostatin is found using the assay system describedmore fully below, it can be sequenced, for example by automated proteinsequencing methods. Alternatively, once the gene or DNA sequence whichcodes for angiostatin is isolated, for example by the methods describedabove, the DNA sequence can be determined using manual or automatedsequencing methods well know in the art. The nucleic acid sequence inturn provides information regarding the amino acid sequence. Thus, ifthe biologically active fragment is generated by specific methods, suchas tryptic digests, or if the fragment is N-terminal sequenced, theremaining amino acid sequence can be determined from the correspondingDNA sequence.

Once the amino acid sequence of the protein is known, the fragment canbe synthesized by techniques well known in the art, as exemplified by“Solid Phase Protein Synthesis: A Practical Approach” E. Atherton and R.C. Sheppard, IRL Press, Oxford, England. Similarly, multiple fragmentscan be synthesized which are subsequently linked together to form largerfragments. These synthetic protein fragments can also be made with aminoacid substitutions at specific locations to test for agonistic andantagonistic activity in vitro and in vivo. Protein fragments thatpossess high affinity binding to tissues can be used to isolate theangiostatin receptor on affinity columns. Isolation and purification ofthe angiostatin receptor is a fundamental step towards elucidating themechanism of action of angiostatin. Isolation of an angiostatin receptorand identification of angiostatin agonists and antagonists willfacilitate development of drugs to modulate the activity of theangiostatin receptor, the final pathway to biological activity.Isolation of the receptor enables the construction of nucleotide probesto monitor the location and synthesis of the receptor, using in situ andsolution hybridization technology. Further, the gene for the angiostatinreceptor can be isolated, incorporated into an expression vector andtransfected into cells, such as patient tumor cells to increase theability of a cell type, tissue or tumor to bind angiostatin and inhibitlocal angiogenesis.

Angiostatin is effective in treating diseases or processes that aremediated by, or involve, angiogenesis. The present invention includesthe method of treating an angiogenesis mediated disease with aneffective amount of angiostatin, or a biologically active fragmentthereof, or combinations of angiostatin fragments that collectivelypossess anti-angiogenic activity, or angiostatin agonists andantagonists. The angiogenesis mediated diseases include, but are notlimited to, solid tumors; blood born tumors such as leukemias; tumormetastasis; benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Angiostatin is useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions, Crohn'sdisease, atherosclerosis, scleroderma, and hypertrophic scars, i.e.,keloids. Angiostatin can be used as a birth control agent by preventingvascularization required for embryo implantation. Angiostatin is usefulin the treatment of diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa) andulcers (Helicobacter pylori).

The synthetic protein fragments of angiostatin have a variety of uses.The protein that binds to the angiostatin receptor with high specificityand avidity is radiolabeled and employed for visualization andquantitation of binding sites using autoradiographic and membranebinding techniques. This application provides important diagnostic andresearch tools. Knowledge of the binding properties of the angiostatinreceptor facilitates investigation of the transduction mechanisms linkedto the receptor.

In addition, labeling angiostatin proteins with short lived isotopesenables visualization of receptor binding sites in vivo using positronemission tomography or other modern radiographic techniques to locatetumors with angiostatin binding sites.

Systematic substitution of amino acids within these synthesized proteinsyields high affinity protein agonists and antagonists to the angiostatinreceptor that enhance or diminish angiostatin binding to its receptor.Such agonists are used to suppress the growth of micrometastases,thereby limiting the spread of cancer. Antagonists to angiostatin areapplied in situations of inadequate vascularization, to block theinhibitory effects of angiostatin and promote angiogenesis. For example,this treatment may have therapeutic effects to promote wound healing indiabetics.

Angiostatin proteins are employed to develop affinity columns forisolation of the angiostatin receptor from cultured tumor cells.Isolation and purification of the angiostatin receptor is followed byamino acid sequencing. Using this information the gene or genes codingfor the angiostatin receptor can be identified and isolated. Next,cloned nucleic acid sequences are developed for insertion into vectorscapable of expressing the receptor. These techniques are well known tothose skilled in the art. Transfection of the nucleic acid sequence(s)coding for angiostatin receptor into tumor cells, and expression of thereceptor by the transfected tumor cells enhances the responsiveness ofthese cells to endogenous or exogenous angiostatin and therebydecreasing the rate of metastatic growth.

Cytotoxic agents such as ricin, are linked to angiostatin, and highaffinity angiostatin protein fragments, thereby providing a tool fordestruction of cells that bind angiostatin. These cells may be found inmany locations, including but not limited to, micrometastases andprimary tumors. Proteins linked to cytotoxic agents are infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity angiostatin fragments are deliveredthrough a cannula into vessels supplying the target site or directlyinto the target. Such agents are also delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae. A combination ofangiostatin antagonists may be co-applied with stimulators ofangiogenesis to increase vascularization of tissue. This therapeuticregimen provides an effective means of destroying metastatic cancer.

Angiostatin may be used in combination with other compositions andprocedures for the treatment of diseases. For example, a tumor may betreated conventionally with surgery, radiation or chemotherapy combinedwith angiostatin and then angiostatin may be subsequently administeredto the patient to extend the dormancy of micrometastases and tostabilize and inhibit the growth of any residual primary tumor.Additionally, angiostatin, angiostatin fragments, angiostatin antisera,angiostatin receptor agonists, angiostatin receptor antagonists, orcombinations thereof, are combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrix, such asbiodegradable polymers, to form therapeutic compositions.

A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polyproteins,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

The angiogenesis-modulating therapeutic composition of the presentinvention may be a solid, liquid or aerosol and may be administered byany known route of administration. Examples of solid therapeuticcompositions include pills, creams, and implantable dosage units. Thepills may be administered orally, the therapeutic creams may beadministered topically. The implantable dosage units may be administeredlocally, for example at a tumor site, or which may be implanted forsystemic release of the therapeutic angiogenesis-modulating composition,for example subcutaneously. Examples of liquid composition includeformulations adapted for injection subcutaneously, intravenously,intraarterially, and formulations for topical and intraocularadministration. Examples of aerosol formulation include inhalerformulation for administration to the lungs.

The angiostatin of the present invention also can be used to generateantibodies that are specific for the inhibitor and its receptor. Theantibodies can be either polyclonal antibodies or monoclonal antibodies.These antibodies that specifically bind to the angiostatin orangiostatin receptors can be used in diagnostic methods and kits thatare well known to those of ordinary skill in the art to detect orquantify the angiostatin or angiostatin receptors in a body fluid ortissue. Results from these tests can be used to diagnose or predict theoccurrence or recurrence of a cancer and other angiogenic mediateddiseases.

The angiostatin also can be used in a diagnostic method and kit todetect and quantify antibodies capable of binding angiostatin. Thesekits would permit detection of circulating angiostatin antibodies whichindicates the spread of micrometastases in the presence of angiostatinsecreted by primary tumors in situ. Patients that have such circulatinganti-angiostatin antibodies may be more likely to develop multipletumors and cancers, and may be more likely to have recurrences of cancerafter treatments or periods of remission. The Fab fragments of theseanti-angiostatin antibodies may be used as antigens to generateanti-angiostatin Fab-fragment antisera which can be used to neutralizeanti-angiostatin antibodies. Such a method would reduce the removal ofcirculating angiostatin by anti-angiostatin antibodies, therebyeffectively elevating circulating angiostatin levels.

Another aspect of the present invention is a method of blocking theaction of excess endogenous angiostatin. This can be done by passivelyimmunizing a human or animal with antibodies specific for the undesiredangiostatin in the system. This treatment can be important in treatingabnormal ovulation, menstruation and placentation, and vasculogenesis.This provides a useful tool to examine the effects of angiostatinremoval on metastatic processes. The Fab fragment of angiostatinantibodies contains the binding site for angiostatin. This fragment isisolated from angiostatin antibodies using techniques known to thoseskilled in the art. The Fab fragments of angiostatin antisera are usedas antigens to generate production of anti-Fab fragment serum. Infusionof this antiserum against the Fab fragments of angiostatin preventsangiostatin from binding to angiostatin antibodies. Therapeutic benefitis obtained by neutralizing endogenous anti-angiostatin antibodies byblocking the binding of angiostatin to the Fab fragments ofanti-angiostatin. The net effect of this treatment is to facilitate theability of endogenous circulating angiostatin to reach target cells,thereby decreasing the spread of metastases.

It is to be understood that the present invention is contemplated toinclude any derivatives of the angiostatin that have endothelialinhibitory activity. The present invention includes the entireangiostatin protein, derivatives of the angiostatin protein andbiologically-active fragments of the angiostatin protein. These includeproteins with angiostatin activity that have amino acid substitutions orhave sugars or other molecules attached to amino acid functional groups.The present invention also includes genes that code for angiostatin andthe angiostatin receptor, and to proteins that are expressed by thosegenes.

The proteins and protein fragments with the angiostatin activitydescribed above can be provided as isolated and substantially purifiedproteins and protein fragments in pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal, intraperitoneal, intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.In addition, the angiostatin may be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor or implanted so that the angiostatin isslowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of angiostatinthrough cannulae to the site of interest, such as directly into ametastatic growth or into the vascular supply to that tumor. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al., J. Neurosurg. 74:441-446 (1991), which is herebyincorporated by reference in its entirety.

The dosage of the angiostatin of the present invention will depend onthe disease state or condition being treated and other clinical factorssuch as weight and condition of the human or animal and the route ofadministration of the compound. For treating humans or animals, betweenapproximately 0.5 mg/kilogram to 500 mg/kilogram of the angiostatin canbe administered. Depending upon the half-life of the angiostatin in theparticular animal or human, the angiostatin can be administered betweenseveral times per day to once a week. It is to be understood that thepresent invention has application for both human and veterinary use. Themethods of the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time.

The angiostatin formulations include those suitable for oral, rectal,ophthalmic (including intravitreal or intracameral), nasal, topical(including buccal and sublingual), intrauterine, vaginal or parenteral(including subcutaneous, intraperitoneal, intramuscular, intravenous,intradermal, intracranial, intratracheal, and epidural) administration.The angiostatin formulations may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients, particularly mentioned above, the formulations of thepresent invention may include other agents conventional in the arthaving regard to the type of formulation in question. Optionally,cytotoxic agents may be incorporated or otherwise combined withangiostatin proteins, or biologically functional protein fragmentsthereof, to provide dual therapy to the patient.

Angiogenesis inhibiting proteins of the present invention can besynthesized in a standard microchemical facility and purity checked withHPLC and mass spectrophotometry. Methods of protein synthesis, HPLCpurification and mass spectrophotometry are commonly known to thoseskilled in these arts. Angiostatin proteins and angiostatin receptorsproteins are also produced in recombinant E. coli or yeast expressionsystems, and purified with column chromatography.

Different protein fragments of the intact angiostatin molecule can besynthesized for use in several applications including, but not limitedto the following; as antigens for the development of specific antisera,as agonists and antagonists active at angiostatin binding sites, asproteins to be linked to, or used in combination with, cytotoxic agentsfor targeted killing of cells that bind angiostatin. The amino acidsequences that comprise these proteins are selected on the basis oftheir position on the exterior regions of the molecule and areaccessible for binding to antisera. The amino and carboxyl termini ofangiostatin, as well as the mid-region of the molecule are representedseparately among the fragments to be synthesized.

These protein sequences are compared to known sequences using proteinsequence databases such as GenBank, Brookhaven Protein, SWISS-PROT, andPIR to determine potential sequence homologies. This informationfacilitates elimination of sequences that exhibit a high degree ofsequence homology to other molecules, thereby enhancing the potentialfor high specificity in the development of antisera, agonists andantagonists to angiostatin.

Angiostatin and angiostatin derived proteins can be coupled to othermolecules using standard methods. The amino and carboxyl termini ofangiostatin both contain tyrosine and lysine residues and areisotopically and nonisotopically labeled with many techniques, forexample radiolabeling using conventional techniques (tyrosineresidues-chloramine T, iodogen, lactoperoxidase; lysineresidues-Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. Alternatively, tyrosine or lysine isadded to fragments that do not have these residues to facilitatelabeling of reactive amino and hydroxyl groups on the protein. Thecoupling technique is chosen on the basis of the functional groupsavailable on the amino acids including, but not limited to amino,sulfhydral, carboxyl, amide, phenol, and imidazole. Various reagentsused to effect these couplings include among others, glutaraldehyde,diazotized benzidine, carbodiimide, and p-benzoquinone.

Angiostatin proteins are chemically coupled to isotopes, enzymes,carrier proteins, cytotoxic agents, fluorescent molecules,chemiluminescent, bioluminescent and other compounds for a variety ofapplications. The efficiency of the coupling reaction is determinedusing different techniques appropriate for the specific reaction. Forexample, radiolabeling of an angiostatin protein with ¹²⁵I isaccomplished using chloramine T and Na¹²⁵I of high specific activity.The reaction is terminated with sodium metabisulfite and the mixture isdesalted on disposable columns. The labeled protein is eluted from thecolumn and fractions are collected. Aliquots are removed from eachfraction and radioactivity measured in a gamma counter. In this manner,the unreacted Na¹²⁵I is separated from the labeled angiostatin protein.The protein fractions with the highest specific radioactivity are storedfor subsequent use such as analysis of the ability to bind toangiostatin antisera.

Another application of protein conjugation is for production ofpolyclonal antisera. For example, angiostatin proteins containing lysineresidues are linked to purified bovine serum albumin usingglutaraldehyde. The efficiency of the reaction is determined bymeasuring the incorporation of radiolabeled protein. Unreactedglutaraldehyde and protein are separated by dialysis. The conjugate isstored for subsequent use.

Antiserum against angiostatin, angiostatin analogs, protein fragments ofangiostatin and the angiostatin receptor can be generated. After proteinsynthesis and purification, both monoclonal and polyclonal antisera areraised using established techniques known to those skilled in the art.For example, polyclonal antisera may be raised in rabbits, sheep, goatsor other animals. Angiostatin proteins conjugated to a carrier moleculesuch as bovine serum albumin, or angiostatin itself, is combined with anadjuvant mixture, emulsified and injected subcutaneously at multiplesites on the back, neck, flanks, and sometimes in the footpads. Boosterinjections are made at regular intervals, such as every 2 to 4 weeks.Blood samples are obtained by venipuncture, for example using themarginal ear veins after dilation, approximately 7 to 10 days after eachinjection. The blood samples are allowed to clot overnight at 4 C andare centrifuged at approximately 2400×g at 4 C for about 30 minutes. Theserum is removed, aliquoted, and stored at 4 C for immediate use or at−20 to −90 C for subsequent analysis.

All serum samples from generation of polyclonal antisera or mediasamples from production of monoclonal antisera are analyzed fordetermination of antibody titer. Titer is established through severalmeans, for example, using dot blots and density analysis, and also withprecipitation of radiolabeled protein-antibody complexes using proteinA, secondary antisera, cold ethanol or charcoal-dextran followed byactivity measurement with a gamma counter. The highest titer antiseraare also purified on affinity columns which are commercially available.Angiostatin proteins are coupled to the gel in the affinity column.Antiserum samples are passed through the column and anti-angiostatinantibodies remain bound to the column. These antibodies are subsequentlyeluted, collected and evaluated for determination of titer andspecificity.

The highest titer angiostatin antisera is tested to establish thefollowing; a) optimal antiserum dilution for highest specific binding ofthe antigen and lowest non-specific binding, b) the ability to bindincreasing amounts of angiostatin protein in a standard displacementcurve, c) potential cross-reactivity with related proteins and proteins,including plasminogen and also angiostatin of related species, d)ability to detect angiostatin proteins in extracts of plasma, urine,tissues, and in cell culture media.

Kits for measurement of angiostatin, and the angiostatin receptor, arealso contemplated as part of the present invention. Antisera thatpossess the highest titer and specificity and can detect angiostatinproteins in extracts of plasma, urine, tissues, and in cell culturemedia are further examined to establish easy to use kits for rapid,reliable, sensitive, and specific measurement and localization ofangiostatin. These assay kits include but are not limited to thefollowing techniques; competitive and non-competitive assays,radioimmunoassay, bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISA, microtiter plates, antibodycoated strips or dipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

One example of an assay kit commonly used in research and in the clinicis a radioimmunoassay (RIA) kit. An angiostatin RIA is illustratedbelow. After successful radioiodination and purification of angiostatinor an angiostatin protein, the antiserum possessing the highest titer isadded at several dilutions to tubes containing a relatively constantamount of radioactivity, such as 10,000 cpm, in a suitable buffersystem. Other tubes contain buffer or preimmune serum to determine thenon-specific binding. After incubation at 4 C for 24 hours, protein A isadded and the tubes are vortexed, incubated at room temperature for 90minutes, and centrifuged at approximately 2000-2500×g at 4 C toprecipitate the complexes of antibody bound to labeled antigen. Thesupernatant is removed by aspiration and the radioactivity in thepellets counted in a gamma counter. The antiserum dilution that bindsapproximately 10 to 40% of the labeled protein after subtraction of thenon-specific binding is further characterized.

Next, a dilution range (approximately 0.1 pg to 10 ng) of theangiostatin protein used for development of the antiserum is evaluatedby adding known amounts of the protein to tubes containing radiolabeledprotein and antiserum. After an additional incubation period, forexample, 24 to 48 hours, protein A is added and the tubes centrifuged,supernatant removed and the radioactivity in the pellet counted. Thedisplacement of the binding of radiolabeled angiostatin protein by theunlabeled angiostatin protein (standard) provides a standard curve.Several concentrations of other angiostatin protein fragments,plasminogen, angiostatin from different species, and homologous proteinsare added to the assay tubes to characterize the specificity of theangiostatin antiserum.

Extracts of various tissues, including but not limited to primary andsecondary tumors, Lewis lung carcinoma, cultures of angiostatinproducing cells, placenta, uterus, and other tissues such as brain,liver, and intestine, are prepared using extraction techniques that havebeen successfully employed to extract angiostatin. After lyophilizationor Speed Vac of the tissue extracts, assay buffer is added and differentaliquots are placed into the RIA tubes. Extracts of known angiostatinproducing cells produce displacement curves that are parallel to thestandard curve, whereas extracts of tissues that do not produceangiostatin do not displace radiolabeled angiostatin from theangiostatin antiserum. In addition, extracts of urine, plasma, andcerebrospinal fluid from animals with Lewis lung carcinoma are added tothe assay tubes in increasing amounts. Parallel displacement curvesindicate the utility of the angiostatin assay to measure angiostatin intissues and body fluids.

Tissue extracts that contain angiostatin are additionally characterizedby subjecting aliquots to reverse phase HPLC. Eluate fractions arecollected, dried in Speed Vac, reconstituted in RIA buffer and analyzedin the angiostatin RIA. The maximal amount of angiostatinimmunoreactivity is located in the fractions corresponding to theelution position of angiostatin.

The assay kit provides instructions, antiserum, angiostatin orangiostatin protein, and possibly radiolabeled angiostatin and/orreagents for precipitation of bound angiostatin-angiostatin antibodycomplexes. The kit is useful for the measurement of angiostatin inbiological fluids and tissue extracts of animals and humans with andwithout tumors.

Another kit is used for localization of angiostatin in tissues andcells. This angiostatin immunohistochemistry kit provides instructions,angiostatin antiserum, and possibly blocking serum and secondaryantiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This angiostatin immunohistochemistry kit permitslocalization of angiostatin in tissue sections and cultured cells usingboth light and electron microscopy. It is used for both research andclinical purposes. For example, tumors are biopsied or collected andtissue sections cut with a microtome to examine sites of angiostatinproduction. Such information is useful for diagnostic and possiblytherapeutic purposes in the detection and treatment of cancer. Anothermethod to visualize sites of angiostatin biosynthesis involvesradiolabeling nucleic acids for use in in situ hybridization to probefor angiostatin messenger RNA. Similarly, the angiostatin receptor canbe localized, visualized and quantitated with immunohistochemistrytechniques.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1 Choice of an Animal-Tumor System in which Growth of Metastasisis Inhibited by the Primary Tumor and is Accelerated after Removal ofthe Primary Tumor

By screening a variety of murine tumors capable of inhibiting their ownmetastases, a Lewis lung carcinoma was selected in which the primarytumor most efficiently inhibited lung metastasis. Syngeneic C57BI6/Jsix-week-old male mice were injected (subcutaneous dorsum) with 1×10⁶tumor cells. Visible tumors first appeared after 3-4 days. When tumorswere approximately 1500 mm³ in size, mice were randomized into twogroups. The primary tumor was completely excised in the first group andleft intact in the second group after a sham operation. Although tumorsfrom 500 mm³ to 3000 mm³ inhibited growth of metastases, 1500 mm³ wasthe largest primary tumor that could be safely resected with highsurvival and no local recurrence.

After 21 days, all mice were sacrificed and autopsied. In mice with anintact primary tumor, there were four +2 visible metastases, compared tofifty +5 metastases in the mice in which the tumor had been removed(p<0.0001). These data were confirmed by lung weight, which correlatesclosely with tumor burden, as has been previously demonstrated. Therewas a 400% increase in wet lung weight in the mice that had their tumorsremoved compared to mice in which the tumor remained intact (p<0.0001).

This experimental model gave reproducible data and the experimentdescribed is reproducible. This tumor is labeled “Lewis lungcarcinoma-low metastatic” (LLC-Low). The tumor also suppressedmetastases in a nearly identical pattern in SCID mice, which aredeficient in both B and T lymphocytes.

EXAMPLE 2 Isolation of a Variant of Lewis Lung Carcinoma Tumor that isHighly Metastatic, Whether or not the Primary Tumor is Removed

A highly metastatic variant of Lewis lung carcinoma arose spontaneouslyfrom the LLC-Low cell line of Example 1 in one group of mice and hasbeen isolated according to the methods described in Example 1 andrepeatedly transplanted. This tumor (LLC-High) forms more than 30visible lung metastases whether or not the primary tumor is present.

EXAMPLE 3 Size of Metastases and Proliferation Rate of Tumor Cellswithin them. Effect of the Primary Tumor that Inhibits Metastases(LLC-Low)

C57BI6/J mice were used in all experiments. Mice were inoculatedsubcutaneously with LLC-Low cells, and 14 days later the primary tumorwas removed in half of the mice. At 5, 10 and 15 days after the tumorhad been removed, mice were sacrificed. Histological sections of lungmetastases were obtained. Mice with an intact primary tumor hadmicrometastases in the lung which were not neovascularized. Thesemetastases were restricted to a diameter of 12-15 cell layers and didnot show a significant size increase even 15 days after tumor removal.In contrast, animals from which the primary tumor was removed, revealedlarge vascularized metastases as early as 5 days after operation. Thesemetastases underwent a further 4-fold increase in volume by the 15th dayafter the tumor was removed (as reflected by lung weight and histology).Approximately 50% of the animals who had a primary tumor removed died oflung metastases before the end of the experiment. All animals with anintact primary tumor survived to the end of the experiment.

Replication rate of tumor cells within metastases was determined bycounting nuclei stained with BrdU which had been previously injectedinto the mice. The high percentage of tumor cells incorporating BrdU insmall, avascular metastases of animals with an intact primary tumor wasequivalent to the BrdU incorporation of tumor cells in the largevascularized metastases of mice from which the primary tumor had beenremoved (FIG. 3). This finding suggests that the presence of a primarytumor has no direct effect on the replication rate of tumor cells withina metastasis.

In FIG. 3, the left panel shows BrdU labeling index of tumor cells inthe lung in the presence or absence of a primary tumor. Beforeimmunohistochemical staining, sections were permeabilized with 0.2 M HClfor 10 minutes and digested with 1 μg/ml proteinase K (BoehringerMannheim GmbH, Mannheim, Germany) in 0.2 M Tris-HCl, 2 mM CaCl₂ at 37°C. for 15 minutes. Labeling index was estimated by counting percentageof positive nuclei at 250 power. The right panel of FIG. 3 depicts ananalysis of total lung weight of tumors with primary tumors intact orremoved 5, 10 and 15 days after operation. Animals were sacrificed 6hours after intraperitoneal injection of BrdU (0.75 mg/mouse).

EXAMPLE 4 Inhibition of Angiogenesis in Lung Metastases in the Presenceof an Intact Primary Tumor

To measure the degree of vascularization in lung metastases, tissueswere stained with antibodies against von Willebrand factor (anendothelial specific marker, available from Dako Inc., Carpenteria,Calif.). Metastases from animals with intact tumors formed a thin cuff(8-12 tumor cell layers) around existing pulmonary vessels. Except forthe endothelial cells of the vessel lining, no or few cells werepositive for von Willebrand factor. In contrast, lung metastases ofanimals 5 days after removal of the primary tumor were not only largerbut were also infiltrated with capillary sprouts containing endothelialcells which stained strongly for von Willebrand factor.

In immunohistochemical analysis of the presence of endothelial cells inlung metastases, a lung metastasis with the primary lung tumor intact 19days after inoculation, had a cuff of tumor cells around a pre-existingmicrovessel in the lung. The metastasis was limited to 8 to 12 celllayers. There was no evidence of neovascularization around themicrovessel, and it did not contain any new microvessels. This wastypical of the maximum size of an avascular pre-angiogenic metastasis.

In an immunohistochemical analysis of tissue collected five days afterthe primary tumor was resected (19 days after inoculation of the primarytumor), the metastasis surrounded a pre-existing vessel in the lung. Incontrast, in the sample where the primary tumor was not resected, thetumor was neovascularized. Thus, an intact primary tumor inhibitsformation of new capillary blood vessels in metastases, butproliferation of tumor cells within a metastasis are not affected by theprimary tumor.

EXAMPLE 5 A Primary Tumor Inhibits Angiogenesis of a Second TumorImplanted in the Mouse Cornea. Growth of this Second Tumor is Inhibited

A 0.25 to 0.5 mm² Lewis lung tumor (LLC-Low) was implanted in the mousecornea on day 0. (Muthukkaruppan Vr., et al., Angiogenesis in the mousecornea. Science 205:1416-1418, 1979) A primary tumor was formed byinoculating 1×10⁶ LLC-Low cells subcutaneously in the dorsum, either 4or 7 days before the corneal implant; or on the day of the cornealimplant; or 4 or 7 days after the corneal implant. Control mice receivedthe corneal implant but not the subcutaneous tumor. Other control micereceived the corneal implant and an inoculation of LLC-High tumor cellsin the dorsum 4 days before the corneal implant. The corneas wereevaluated daily by slit-lamp stereomicroscopy for the growth of thecorneal tumor (measured by an ocular micrometer) and for the growth ofnew capillary vessels from the edge of the corneal limbus.

In control mice not bearing a primary subcutaneous tumor, a majority ofcorneas (6/8) developed neovascularization starting at day 6 to 7 daysafter corneal implantation and continuing to day 10. By day 10, thevascularized corneal tumors had reached approximately a quarter of thevolume of the whole eye. In the presence of the primary subcutaneousLLC-Low tumor, the corneal implants did not become vascularized if theprimary tumor was in place by at least 4 days or more before the cornealimplant (Table 1). In the absence of neovascularization, corneal tumorsgrew slowly as thin, white, avascular discs within the cornea.

However, if the primary tumor was not implanted until 4 days after thecorneal implant, corneas became vascularized and 3/3 corneal tumors grewat similar rates as the non-tumor bearing controls. In the presence ofthe primary subcutaneous LLC-High tumor, the majority of corneas (2/3)developed neovascularization starting at day 7 after cornealimplantation and continuing to day 10. By day 10, the vascularizedcorneal tumors again had reached approximately a quarter of the volumeof the whole eye.

TABLE 1 Inhibition of tumor angiogenesis in the cornea by a primarysubcutaneous tumor. Day of eye implant 0 0 0 0 0 0 0 Day of primarytumor −7 −4 −4 * 0 none +4 +7 implant Number of mice with new 2/10 0/92/3 2/3 6/8 3/3 2/3 corneal vessels at day 10 [All primary tumors areLLC-Low except (*) which is LLC-High].It would be expected that 0/10 corneas would show neovascularizationwhen the primary LLC-Low subcutaneous tumor was implanted 7 days beforethe eye tumor implant (i.e. −7). However, 2 of the tumors (2/10) hadbecome necrotic because they were too large (>3 cm³).

EXAMPLE 6 Primary Intact Tumor Inhibits Angiogenesis Induced by aSecondary Subcutaneous Implant of Basic Fibroblast Growth Factor (bFGF.)

Although the experiments described in Examples V and VI show that aprimary tumor inhibits angiogenesis in a secondary metastasis, thesestudies do not reveal whether the primary tumor: (i) inhibitsendothelial proliferation (or angiogenesis) directly, or (ii) indirectlyby down-regulating the angiogenic activity of the metastatic tumorcells. To distinguish between these two possibilities, a focus ofsubcutaneous angiogenesis was induced by an implant of matrigelcontaining basic fibroblast growth factor (bFGF). (Passaniti A, et al.,A simple, quantitative method for assessing angiogenesis andanti-angiogenic agents using reconstituted basement membrane, heparinand fibroblast growth factor. Lab. Invest. 67:519, 1992)

Matrigel (an extract of basement membrane proteins), containing either25 or 50 ng/ml bFGF in the presence of heparin, was injectedsubcutaneously on the ventral surface of normal and tumor-bearing mice(LLC-Low). Mice were sacrificed 4 days later and hemoglobinconcentration in the gel was measured to quantify blood vesselformation. It has previously been shown that the number of new vesselswhich enter the matrigel is correlated with hemoglobin concentration.(Folkman J., Angiogenesis and its inhibitors in “Important Advances inOncology 1985”, V T DeVita, S. Hellman and S. Rosenberg, editors, J.B.Lippincott, Philadelphia 1985) Some gels were also prepared forhistological examination. In normal mice, matrigel pellets whichcontained 50 ng/ml bFGF were completely red. They were heavily invadedby new capillary vessels, and contained 2.4 g/dl hemoglobin. Matrigelwhich lacked bFGF was translucent and gray and contained only 0.4 g/dlhemoglobin (a 6-fold difference). In contrast, matrigel from mice with aprimary tumor contained only 0.5 g/dl (FIG. 4).

The near complete inhibition of angiogenesis in this experiment suggeststhat the presence of a Lewis lung primary tumor can inhibit bFGF-inducedangiogenesis directly.

EXAMPLE 7 Transfer of Serum from a Tumor-Bearing Animal to an Animalfrom which the Primary Tumor has been Removed Suppresses Metastases

Mice were implanted with Lewis lung carcinoma as described above. After15 days, when tumors were approximately 1500 mm³, the mice wererandomized into four groups. Three groups underwent complete surgicalresection of the primary tumor; in one group the tumors were left inplace (after a sham surgical procedure). The mice in the three resectiongroups then received daily intraperitoneal injections of saline, serumfrom normal nontumor bearing mice, or serum from mice with 1500 mm³Lewis lung carcinomas. The group of mice with the tumors left intactreceived intraperitoneal saline injections. All mice were treated for 21days, after which the animals were euthanized and lung metastases werecounted (Table 2).

TABLE 2 Primary Primary Tumor Removed Tumor Treatment Serum from Intact(Intraperitoneal Serum from tumor-bearing Saline Injections) Salinenormal mice mice Injections Number of Lung 55 ± 15 50 ± 4 7 ± 2 3 ± 1Metastases:These results were confirmed by lung weight. p=<0.0001 for thedifference between the two groups [(55 & 50) vs. (7 & 3)]. Similarresults have been obtained using angiostatin from the urine oftumor-bearing animals.

EXAMPLE 8 Bovine Capillary Endothelial (BCE) Cell Assay

BCE cells are used between passages 9 and 14 only. At day 0, BCE cellsare plated onto gelatinized (1.5% gelatin in PBS at 37°, 10% CO₂ for 24hours and then rinsed with 0.5 ml PBS) 24 well plates at a concentrationof 12,500 cells/well. Cell counts are performed using a hemocytometer.Cells are plated in 500 μl DMEM with 10% heat-inactivated (56° C. for 20minutes) bovine calf serum and 1% glutamine-pen-strep (GPS).

BCE cells are challenged as follows: Media is removed and replaced with250 μl of DMEM/5% BCS/1% GPS. The sample to be tested is then added towells. (The amount varies depending on the sample being tested) Platesare placed at 37° C./10% CO₂ for approximately 10 minutes. 250 μl ofDMEM/5% BCS/1% GPS with 2 ng/ml bFGF is added to each well. The finalmedia is 500 μl of DMEM/5% BCS/1% GPS/ with 1 ng/ml bFGF. The plate isreturned to 37° C./10% CO₂ incubator for 72 hours.

At day 4, cells are counted by removing the medium and then trypsinizingall wells (0.5 ml trypsin/EDTA) for 2 to 3 minutes. The suspended cellsare then transferred to scintillation vials with 9.5 ml Hemetall andcounted using a Coulter counter. A unit of activity is that amount ofserum containing angiostatin that is capable of producing half-maximalinhibition of capillary endothelial proliferation when endothelial cellsare incubated in bFGF 1 ng/ml for 72 hours.

EXAMPLE 9 Serum from Mice Bearing the Low Metastatic Lewis Lung Tumor(LLC-Low) Inhibits Capillary Endothelial Cell Proliferation In Vitro

Bovine capillary endothelial cells were stimulated by basic fibroblastgrowth factor (bFGF 1 ng/ml), in a 72-hour proliferation assay. Theserum of tumor-bearing mice added to these cultures inhibitedendothelial cell proliferation in a dose-dependent and reversiblemanner. Normal serum was not inhibitory (FIG. 5). Endothelial cellproliferation was inhibited in a similar manner (relative to controls)by serum obtained from tumor-bearing nu/nu mice and SCID mice. After theprimary tumor was removed, angiostatin activity disappeared from theserum by 3-5 days.

Tumor-bearing serum also inhibited bovine aortic endothelial cells andendothelial cells derived from a spontaneous mouse hemangioendothelioma,(Obeso, et al., “Methods in Laboratory Investigation, AHemangioendothelioma-derived cell line; Its use as a Model for the Studyof Endothelial Cell Biology,” Lab Invest., 63(2), pgs 259-269, 1990) butdid not inhibit Lewis lung tumor cells, 3T3 fibroblasts, aortic smoothmuscle cells, mink lung epithelium, or W138 human fetal lungfibroblasts.

EXAMPLE 10 Serum from Mice Bearing the Lewis Lung Tumor (LLC-High) thatdoes not Inhibit Metastases, does not Inhibit Capillary Endothelial CellProliferation In Vitro

Serum from mice bearing a primary tumor of the LLC-High did notsignificantly inhibit proliferation of bFGF-stimulated bovine capillaryendothelial cells relative to controls. Also, when this serum wassubjected to the first two steps of purification (heparin-Sepharosechromatography and gel filtration), angiostatin activity was not foundin any fractions.

EXAMPLE 11 Ascites from Lewis Lung Carcinoma (Low Metastatic), AlsoGenerates Angiostatin Serum

Mice received intraperitoneal injections of either LLC-Low or LLC-Hightumor cells (106), and one week later, 1-2 ml of bloody ascites wasobtained from each of 10-20 mice. Mesenteric tumor seeding was seen. Themice were then euthanized. Serum was obtained by cardiac puncture. Serumwas also obtained from normal, non-tumor-bearing mice as a control.Serum and ascites were centrifuged to remove cells, and the supernatewas assayed on bovine capillary endothelial cells stimulated by bFGF (1ng/ml) (see Example IX). Ascites originating from both tumor typesstimulated significant proliferation of capillary endothelial cells(e.g., 100% proliferation) over controls after 72 hours (FIG. 6). Incontrast, serum from the low metastatic mice inhibited endothelial cellproliferation (inhibition to 79% of controls). The serum from the highmetastatic line was stimulatory by 200%.

These data show that the ascites of the low metastatic line contains apredominance of endothelial growth stimulator over angiostatin. Thiscondition is analogous to a solid primary tumor. Furthermore,angiostatin activity appears in the serum, as though it were unopposedby stimulatory activity. This pattern is similar to the solid primarytumor (LLC-Low). The ascites from the high metastatic tumor (LLC-High)also appears to contain a predominance of endothelial cell stimulator,but angiostatin cannot be identified in the serum.

EXAMPLE 12 Fractionation of Angiostatin from Serum by ColumnChromatography and Analysis of Growth-Inhibitory Fractions by SDS-PAGE

To purify the angiostatin(s), serum was pooled from tumor-bearing mice.The inhibitory activity, assayed according the above-described in vitroinhibitor activity assay, was sequentially chromatographed usingheparin-Sepharose, Biogel AO.5 mm agarose, and several cycles ofC4-reverse phase high performance liquid chromatography (HPLC). SDS-PAGEof the HPLC fraction which contained endothelial inhibitory activity,revealed a discrete band of apparent reduced M_(r) of 38,000 Daltons,which was purified approximately 1 million-fold (see Table 3) to aspecific activity of approximately 2×10⁷. At different stages of thepurification, pooled fractions were tested with specific antibodies forthe presence of known endothelial inhibitors. Platelet factor-4,thrombospondin, or transforming growth factor beta, were not found inthe partially purified or purified fractions.

TABLE 3 Specific activity (units*/mg) Fold purification Serum 1.69 1Heparin Sepharose 14.92 8.8 Bio-gel AO.5m 69.96 41.4 HPLC/C4 2 × 10⁷ 1.2× 10⁶ *A unit of activity is that amount of serum containing angiostatinthat is capable of producing half-maximal inhibition of capillaryendothelial proliferation when endothelial cells are incubated in bFGF 1ng/ml for 72 hours.

EXAMPLE 13 Fractionation of Angiostatin from Urine by ColumnChromatography and Analysis of Growth-Inhibitory Fractions by SDS-PAGE

Purification of the endothelial cell inhibitor(s) from serum is hamperedby the small volume of serum that can be obtained from each mouse and bythe large amount of protein in the serum.

Urine from tumor bearing mice was analyzed and found that it contains aninhibitor of endothelial cell proliferation that is absent from theurine of non-tumor bearing mice and from mice with LLC-high tumors.Purification of the endothelial cell inhibitory activity was carried outby the same strategy that was employed for purification of serum(described above) (FIG. 7).

FIG. 7 shows C4 reverse phase chromatography of partially purified serumor urine from tumor-bearing animals. All fractions were assayed onbovine capillary endothelial cells with bFGF in a 72-hour proliferationassay as described in Example IX. A discrete peak of inhibition was seenin both cases eluting at 30-35% acetonitrile in fraction 23.SDS-polyacrylamide gel electrophoresis of inhibitory fraction from thethird cycle of C4 reverse phase chromatography of serum fromtumor-bearing animals showed a single band at about 38,000 Daltons.

EXAMPLE 14 Characterization of Circulating Angiostatin

Endothelial inhibition was assayed according to the procedure describedin Example 9. Angiostatin was isolated on a Synchropak HPLC C4 column.(Synchrom, Inc. Lafayette, Ind.) The inhibitor was eluted at 30 to 35%acetonitrile gradient. On a sodium dodecyl sulfate polyacrylamide gelelectrophoresis (PAGE) gel under reducing conditions (b-mercaptoethanol(5% v/v), the protein band with activity eluted at 38 kilodaltons. Undernon-reducing conditions, the protein with activity eluted at 28kilodaltons. The activity is found at similar points whether the initialsample was isolated from urine or from serum. Activity was not detectedwith any other bands.

Activity associated with the bands was lost when heated (100° C. for 10minutes) or treated with trypsin. When the band with activity wasextracted with a water/chloroform mixture (1:1), the activity was foundin the aqueous phase only.

EXAMPLE 15 Purification of Inhibitory Fragments from Human Plasminogen

Plasminogen lysine binding site I was obtained from Sigma ChemicalCompany. The preparation is purified human plasminogen after digestionwith elastase. Lysine binding site I obtained in this manner is apopulation of proteins that contain, in aggregate, at least the firstthree triple-loop structures (numbers 1 through 3) in the plasminA-chain (Kringle 1+2+3). (Sotrrup-Jensen, L., et al. in Progress inChemical Fibrinolysis and Thrombolysis, Vol. 3, 191, Davidson, J. F., etal. eds. Raven Press, New York 1978 and Wiman, B., et al., Biochemica etBiophysica Acta, 579, 142 (1979)). Plasminogen lysine binding site I(Sigma Chemical Company, St. Louis, Mo.) was resuspended in water andapplied to a C4-reversed phase column that had been equilibrated withHPLC-grade water/0.1% TFA. The column was eluted with a gradient ofwater/0.1% TFA to acetonitrile/0.1% TFA and fractions were collectedinto polypropylene tubes. An aliquot of each was evaporated in a speedvac, resuspended with water, and applied to BCEs in a proliferationassay. This procedure was repeated two times for the inhibitoryfractions using a similar gradient for elution. The inhibitory activityeluted at 30-35% acetonitrile in the final run of the C4 column.SDS-PAGE of the inhibitory fraction revealed 3 discrete bands ofapparent reduced molecular mass of 40, 42.5, and 45 kd. SDS-PAGE undernon-reducing conditions revealed three bands of molecular mass 30, 32.5,and 35 kd respectively.

EXAMPLE 16 Extraction of Inhibitory Activity from SDS-PAGE

Purified inhibitory fractions from human plasminogen based purificationswere resolved by SDS-PAGE under non-denaturing conditions. Areas of thegel corresponding to bands seen in neighboring lanes loaded with thesame samples by silver staining were cut from the gel and incubated in 1ml of phosphate buffered saline at 4° C. for 12 hours in polypropylenetubes. The supernatant was removed and dialyzed twice against saline for6 hours (MWCO=6-8000) and twice against distilled water for 6 hours. Thedialysate was evaporated by vacuum centrifugation. The product wasresuspended in saline and applied to bovine capillary endothelial cellsstimulated by 1 ng/ml basic fibroblast growth factor in a 72 hour assay.Protein extracted from each of the three bands inhibited the capillaryendothelial cells.

EXAMPLE 17 Plasminogen Fragment Treatment Studies

Mice were implanted with Lewis lung carcinomas and underwent resectionswhen the tumors were 1500-2000 mm³. On the day of operation, mice wererandomized into 6 groups of 6 mice each. The mice received dailyintraperitoneal injections with the three purified inhibitory fragmentsof human plasminogen, whole human plasminogen, urine from tumor-bearinganimals, urine from normal mice, or saline. One group of tumor-bearinganimals that had only a sham procedure was treated with salineinjections. Immediately after removal of the primary tumor, the micereceive an intraperitoneal injection of 24 μg (1.2 mg/kg/day/mouse) ofthe inhibitory plasminogen fragments as a loading dose. They thenreceive a daily intraperitoneal injections of 12 μg of the inhibitoryfragment (0.6 mg/kg/day/mouse) for the duration of the experiment.Control mice receive the same dose of the whole plasminogen moleculeafter tumor removal. For the urine treatments, the urine of normal ortumor bearing mice is filtered, dialyzed extensively, lyophilized, andthen resuspended in sterile water to obtain a 250 fold concentration.The mice are given 0.8 ml of the dialyzed urine concentrate, either fromtumor bearing mice or normal mice, in two intraperitoneal injections onthe day of removal of the primary tumor as a loading dose. They thenreceive daily intraperitoneal injections of 0.4 ml of the dialyzed andconcentrated urine for the course of the experiment. Treatments werecontinued for 13 days at which point all mice were sacrificed andautopsied.

The results of the experiment are shown in FIGS. 8 and 9. FIG. 8 showssurface lung metastases after the 13 day treatment. Surface lungmetastases refers to the number of metastases seen in the lungs of themice at autopsy. A stereomicroscope was used to count the metastases.FIG. 8 shows the mean number of surface lung metastases that was countedand the standard error of the mean. As shown, the group of mice with theprimary tumor present showed no metastases. The mice in which theprimary tumor was resected and were treated with saline showed extensivemetastases. The mice treated with the human derived plasminogen fragmentshowed no metastases. The mice treated with whole plasminogen showedextensive metastases indicating that the whole plasminogen molecule hasno endothelial inhibitory activity. Those mice treated with dialyzed andconcentrated urine from tumor bearing mice showed no metastases. Micetreated with concentrated urine from normal mice showed extensivemetastases. When the weight of the lung was measured, similar resultswere obtained (FIG. 9).

EXAMPLE 18 Amino Acid Sequence of Murine and Human Angiostatin

The amino acid sequence of angiostatin isolated from mouse urine andangiostatin isolated from the human lysine binding site I fragmentpreparation was determined on an Applied Biosystem Model 477A proteinsequencer. Phenylthiohydantoin amino acid fractions were identified withan on-line ABI Model 120A HPLC. The amino acid sequence determined fromthe N-terminal sequence and the tryptic digests of the murine and humanangiostatin indicate that the sequence of the angiostatin is similar tothe sequence beginning at amino acid number 98 of murine plasminogen.Thus, the amino acid sequence of the angiostatin is a moleculecomprising a protein having a molecular weight of between approximately38 kilodaltons and 45 kilodaltons as determined by reducingpolyacrylamide gel electrophoresis and having an amino acid sequencesubstantially similar to that of a murine plasminogen fragment beginningat amino acid number 98 of an intact murine plasminogen molecule. Thebeginning amino acid sequence of the murine angiostatin (SEQ ID NO:2) isshown in FIG. 1. The length of the amino acid sequence may be slightlylonger or shorter than that shown in the FIG. 1.

N terminal amino acid analysis and tryptic digests of the activefraction of human lysine binding site I (See Example 15) show that thesequence of the fraction begins at approximately amino acid 97 or 99 ofhuman plasminogen and the human angiostatin is homologous with themurine angiostatin. The beginning amino acid sequence of the humanangiostatin (starting at amino acid 98) is shown in FIG. 2, (SEQ IDNO:3). The amino acid sequence of murine and human angiostatin iscompared in FIG. 2 to corresponding internal amino acid sequences fromplasminogen of other species including porcine, bovine, and Rhesusmonkey plasminogen, indicating the presence of angiostatin in thosespecies.

EXAMPLE 19 Expression of Human Angiostatin in E. coli

The pTrcHisA vector (Invitrogen) (FIG. 10) was used to obtainhigh-level, regulated transcription from the trc promoter for enhancedtranslation efficiency of eukaryotic genes in E. coli. Angiostatin isexpressed fused to an N-terminal nickel-binding poly-histidine tail forone-step purification using metal affinity resins. The enterokinasecleavage recognition site in the fusion protein allows for subsequentremoval of the N-terminal histidine fusion protein from the purifiedrecombinant protein. The recombinant human angiostatin protein was foundto bind lysine; is cross-reactive with monoclonal antibodies specificfor kringle regions 1, 2 and 3, and inhibits bFGF-driven endothelialcell proliferation in vitro.

To construct the insert, the gene fragment encoding human angiostatin isobtained from human liver mRNA which is reverse transcribed andamplified using the polymerase chain reaction (PCR) and specificprimers. The product of 1131 base pairs encodes amino acids 93 to 470 ofhuman plasminogen. The amplified fragment was cloned into the XhoI/KpnIsite of pTrcHisA, and the resultant construct transformed into XL-1B(available from Stratagene) E. coli host cells. A control clonecontaining the plasmid vector pTrcHisA alone was transformed into XL-1BE. coli host cells as well. This clone is referred to as the vectorcontrol clone. Both clones were purified identically as described below.

Expressing colonies were selected in the following manner. Colony liftsof E. coli transformed with the gene encoding angiostatin were grown onIPTG impregnated nitrocellulose filters and overlaid on an LB agarplate. Following IPTG induction of expression, colonies were lysed onnitrocellulose filters. The nitrocellulose lifts were blocked, rinsedand probed with two separate monoclonal antibodies (mAbs Dcd and Vap;gift of S. G. McCance and F. J. Castellino, University of Notre Dame)which recognize specific conformations of angiostatin. Stronglyexpressing colonies recognized by the mAbs were selected.

To identify the optimal time for maximal expression, cells werecollected at various times before and after IPTG induction and exposedto repeated freeze-thaw cycles, followed by analysis with SDS-PAGE,immunoblotting and probing with mAbs Dcd and Vap.

From these, clone pTrcHisA/HAsH4 was selected. Induction with IPTG wasfor 4 hours after which the cell pellet was collected and resuspended in50 mM Tris pH 8.0, 2 mM EDTA, 5% glycerol and 200 mg/ml lysozyme andstirred for 30 min. at 4° C. The slurry was centrifuged at 14,000 rpmfor 25 min. and the pellet resuspended in 50 mM Tris pH 8.0, 2 mM EDTA,5% glycerol and 0.1% DOC. This suspension was stirred for 1 hr. at 4°C., and then centrifuged at 14,000 rpm for 25 min. The supernatantfraction at this step contains expressed angiostatin. The E. coliexpressed human angiostatin was found to possess the physical propertyof native angiostatin, that is the ability to bind lysine. The E. coliexpressed angiostatin was thus purified over a lysine-sepharose(Pharmacia or Sigma) column in a single step. Elution of angiostatinfrom the column was with 0.2M epsilon-amino-n-caproic acid pH7.5.

Subsequent to these experiments, scale-up 10 L fermentation batches ofclone pTrcHisA/HAsH4 was performed. The cells obtained from thisscaled-up induction were pelleted and resuspended in 50 mM Tris pH7.5,cracked at 10,000 psi thrice chilling at 10° C. in-between passes. Thelysate obtained was clarified by centrifugation at 10,000 rpm for 30 minat 4° C., and expressed angiostatin isolated over lysine-sepharose (FIG.11).

Purified E. coli expressed human angiostatin was dialysed exhaustivelyagainst water and lyophilized. The expressed human angiostatin wasresuspended in media (DMEM, 5% BCS, 1%Gentamycin/penicillin/streptomycin) to an estimated concentration of 3ug/ml, and used in bovine capillary endothelial (BCE) cell assays invitro, as described in EXAMPLE 8, pg. 39. Similarly, the control clonecontaining the vector alone was treated in the identical fashion as theclone pTrcHisA/HAsH4. It was induced with IPTG identically, and thebacterial lysate used to bind lysine, eluted with 0.2 M amino caproicacid, dialysed exhaustively and lyophilized. This control preparationwas resuspended in media also at an estimated concentration of 3 ug/ml.The samples of recombinant angiostatin, and controls were obtained fromdifferent induction and fermentation batches as well as separatepurification runs, and were all coded at EntreMed, Maryland. BCE assayswere performed with these coded samples in a blinded fashion atChildren's Hospital, Boston.

The results of BCE assays of recombinant human angiostatin showed thathuman angiostatin expressed in E. coli inhibited the proliferation ofBCE cells due to bFGF (used at 1 ng/ml) (FIG. 12). The stock recombinantangiostatin in media (at about 3 ug/ml) was used at a 1:5, 1:10 and 1:20dilution. Percent inhibition was calculated as follows:

$1 - \frac{{{number}{\mspace{11mu} \;}{of}\mspace{14mu} {cells}\mspace{14mu} {with}\mspace{14mu} {angiostatin}} - {{number}\mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {at}\mspace{14mu} {day}\mspace{14mu} 0}}{{{number}{\mspace{11mu} \;}{of}\mspace{14mu} {cells}\mspace{14mu} {with}\mspace{14mu} {bFGF}\mspace{14mu} {alone}} - {{number}\mspace{14mu} {of}\mspace{14mu} {cells}\mspace{14mu} {at}\mspace{14mu} {day}\mspace{14mu} 0}}$

The percent inhibition of BCE cell proliferation was comparable orhigher to that of plasminogen derived angiostatin at similarconcentrations. The results from a repeat run of the BCE assay aredepicted in FIG. 13, where at a 1:5 dilution of the stock, recombinantangiostatin gave similar percent inhibitions to those obtained withplasminogen derived angiostatin. FIG. 13 shows the surprising resultthat human recombinant angiostatin protein inhibits over 60%, and asmuch as over 75% of BCE proliferation in culture.

EXAMPLE 20 Angiostatin Maintains Dormancy of Micrometastases byIncreasing the Rate of Apoptosis

Following subcutaneous inoculation of C57 BL6/J mice with Lewis lungcarcinoma cells (1×10⁶), primary tumors of approximately 1.5 cm³developed. Animals were subject to either surgical removal of theprimary tumor or sham surgery. At 5, 10 and 15 days after surgery, micewere sacrificed and their lungs prepared for histological examination.Animals with resected primary tumors showed massive proliferation ofmicrometastases compared to sham operated controls (FIG. 14). Thesechanges were accompanied by a significant increase in lung weight.

Analysis of tumor cell proliferation, as measured by uptake ofbromo-deoxyuridine (BrdU) showed no differences between animals withintact primary tumors or resected tumors at 5, 9 and 13 days, indicatingthat the increase in tumor mass could not be explained by increasedproliferation (FIG. 15). Accordingly, cell death was examined in theseanimals. Apoptosis, a process of cell death that is dependent on changesin gene expression and accounts for elimination of cells duringdevelopment and in rapidly proliferating tissues such as the smallintestine, was examined by immunohistochemically labeling fragmented DNAwith the terminal deoxynucleotidyl transferase (TdT) technique. Theapoptotic index was determined at each time of sacrifice. The removal ofprimary tumors caused a statistically significant increase(approximately 3 to 4 fold) in the apoptotic index at all times examined(FIG. 15).

Supporting evidence was obtained by treating mice with removed primarytumors with an exogenous suppressor of angiogenesis. This substance,TNP-1470 (O-chloroacetylcarbamoyl fumagillol, previously namedAGM-1470), is an analogue of fumagillin with reported anti-angiogenicactivity. Subcutaneous injection of TNP-1470 (30 mg/kg every two days)produced results that were strikingly similar to those described abovefor animals that had intact primary tumors. These animals displayed alower lung weight, equivalent proliferative index and increasedapoptotic index compared to saline-injected controls (FIG. 16).

These data indicate that metastases remain dormant when tumor cellproliferation is balanced by an equivalent rate of cell death. Theremoval of the primary tumor causes a rapid increase in the growth ofmetastases, probably due to the removal of angiogenesis inhibitors(angiostatin) which control metastatic growth by increasing apoptosis intumor cells. These effects are similar to those seen following removalof primary tumors and administration of an exogenous inhibitor ofangiogenesis. Taken together, these data suggest that the primary tumorreleases angiostatin which maintains dormancy of micrometastases.

EXAMPLE 21 Treatment of Primary Tumors with Angiostatin In Vivo

Angiostatin was purified from human plasminogen by limited elastasedigestion as described in Example 15 above. Angiostatin was resuspendedin phosphate-buffered saline for administration into six week old maleC57BI6/J mice. Animals were implanted subcutaneously with 1×10⁶ tumorcells of either the Lewis lung carcinoma or T241 fibrosarcoma. Treatmentwith angiostatin is begun after four days when tumors are 80-160 mm³ insize. Mice received angiostatin injections in either a single injectionof 40 mg/kg or two 80 mg/kg injections via intraperitoneal (ip) orsubcutaneous (sc) routes. Animals were sacrificed at various times aftertreatment extending to 19 days.

Angiostatin, administered at a daily dose of 40 mg/kg ip, produced ahighly significant inhibition of the growth of T241 primary tumors (FIG.17). This inhibitory effect on growth was visibly evident within 2 daysand increased in magnitude throughout the time course of the study. Byday 18, angiostatin-treated mice had tumors that were approximately 38%of the volume of the saline injected controls. This difference wasstatistically significant (p<0.001, Students t-test).

Angiostatin treatment (total dose of 80 mg/kg/day, administered twicedaily at 40 mg/kg ip or sc) also significantly reduced the growth rateof LLC-LM primary tumors (FIG. 17). This inhibitory effect was evidentat 4 days and increased in magnitude at all subsequent times examined.On the last day of the experiment (day 19), angiostatin-treated micepossessed a mean tumor volume that was only 20% of the saline-injectedcontrols which was significantly different (p<0.001 Students t-test).

In another series of experiments angiostatin was administered (50 mg/kgq12 h) to mice implanted with T241 fibrosarcoma, Lewis lung carcinoma(LM) or reticulum cell sarcoma cells. For each tumor cell type, the micereceiving angiostatin had substantially reduced tumor size. FIG. 19demonstrates that for T241 fibrosarcoma, the angiostatin treated micehad mean tumor volumes that were only 15% of the untreated mice at day24. FIG. 20 demonstrates that for Lewis lung carcinoma (LM), theangiostatin treated mice had mean tumor volumes that were only 13% ofthe untreated mice at day 24. FIG. 21 demonstrates that for reticulumsarcoma, the angiostatin treated mice had mean tumor volumes that wereonly 19% of the untreated mice at day 24. The data represent the averageof 4 mice at each time point.

These results demonstrate that angiostatin is an extremely potentinhibitor of the growth of three different primary tumors in vivo.

EXAMPLE 22 Treatment of Human Cell-Derived Primary Tumors in Mice withAngiostatin In Vivo

The effect of angiostatin on two human tumor cell lines, human prostatecarcinoma PC-3 and human breast carcinoma MDA-MB, was studied.Immunodeficient SCID mice were implanted with human tumor cells, and themice treated with 50 mg/kg angiostatin every 12 hours essentially asdescribed in Example 21. The results demonstrate that the angiostatinprotein of the present invention is a potent inhibitor of human tumorcell growth. FIG. 22 shows that for human prostate carcinoma PC-3, theangiostatin treated mice had only 2% of the mean tumor volume comparedto the untreated control mice at day 24. FIG. 23 shows that for humanbreast carcinoma MDA-MB, the angiostatin treated mice had only 8% of themean tumor volume compared to the untreated control mice at day 24.

EXAMPLE 23 Gene Therapy Effect of Transfection of the Angiostatin Geneon Tumor Volume

A 1380 base pair DNA sequence for angiostatin derived from mouseplasminogen cDNA (obtained from American Type Culture Collection(ATCC)), coding for mouse plasminogen amino acids 1-460, was generatedusing PCR and inserted into an expression vector. The expression vectorwas transfected into T241 fibrosarcoma cells and the transfected cellswere implanted into mice. Control mice received either non-transfectedT241 cells, or T241 cells transfected with the vector only (i.e.non-angiostatin expressing transfected cells). Threeangiostatin-expressing transfected cell clones were used in theexperiment. Mean tumor volume determined over time. The results show thesurprising and dramatic reduction in mean tumor volume in mice for theangiostatin-expressing cells clones as compared with the non-transfectedand non-expressing control cells.

The mouse DNA sequence coding for mouse angiostatin protein is derivedfrom mouse plasminogen cDNA. The mouse angiostatin encompasses mouseplasminogen kringle regions 1-4. The schematic for constructing thisclone is shown in FIG. 24.

The mouse angiostatin protein clones were transfected into T241fibrosarcoma cells using the LIPOFECTIN™ transfection system (availablefrom Life Technologies, Gaithersburg, Md.). The LIPOFECTIN™ reagent is a1:1 (w/w) liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA),and diolecoyl phosphotidylethanolamine (DOPE) in membrane filteredwater.

The procedure for transient transfection of cells is as follows:

-   1. T241 cells are grown in 60 cm² tissue culture dishes,    seed≈1-2×10⁵ cells in 2 ml of the appropriate growth medium    supplemented with serum.-   2. Incubate the cells at 37° C. in a CO₂ incubator until the cells    are 40-70% confluent. will usually take 18-24 h, but the time will    vary among cell types. The T241 tumor cells confluency was    approximately 70%.-   3. Prepare the following solutions in 12×75 mm sterile tubes:    -   Solution A: For each transfection, dilute 5 μg of DNA in 100 μl        of serum-free OPTI-MEM I Reduced Serum Medium (available from        Life Technologies) (tissue culture grade deionized water can        also be used).    -   Solution B: For each transfection, dilute 30 μg of LIPOFECTIN in        100 μl OPTI-MEM medium.-   4. Combine the two solutions, mix gently, and incubate at room    temperature for 10-15 min.-   5. Wash cells twice with serum-free medium.-   6. For each transfection, add 0.8 ml serum-free medium to each tube    containing the LIPOFECTIN™ reagent-DNA complexes. Mix gently and    overlay the complex onto cells.-   7. Incubate the cells for approximately 12 h at 37° C. in a CO₂    incubator.-   8. Replace the DNA containing medium with 1 mg/ml selection medium    containing serum and incubate cells at 37° C. in a CO₂ incubator for    a total of 48-72 h.-   9. Assay cell extracts for gene activity at 48-72 h post    transfection.

Transfected cells can be assayed for expression of angiostatin proteinusing angiostatin-specific antibodies. Alternatively, after about 10-14days, G418 resistant colonies appeared in the CMV-angiostatintransfected T241 cells. Also, a number of clones were seen in the vectoralone transfected clones but not in the untransfected clones. The G418resistant clones were selected for their expression of angiostatin,using a immunofluorence method.

Interestingly, the in vitro cell growth T241 cells and Lewis lung cellstransfected with angiostatin was not inhibited or otherwise adverselyaffected, as shown in FIGS. 25 and 26.

FIG. 27 depicts the results of the transfection experiment. All three ofthe angiostatin-expressing T241 transfected clones produced mean tumorvolumes in mice that were substantially reduced relative to the tumorvolume in control mice. The mean tumor volume of the mice implanted withClone 37 was only 13% of the control, while Clone 31 and Clone 25 tumorvolumes were only 21% and 34% of the control tumor volumes,respectively. These results demonstrate that the DNA sequences codingfor angiostatin can be transfected into cells, that the transfected DNAsequences are capable of expressing angiostatin protein by implantedcells, and that the expressed angiostatin functions in vivo to reducetumor growth.

EXAMPLE 24 Localization of In Vivo Site of Angiostatin Expression

To localize the in vivo site of expression of angiostatin protein, totalRNA from various cell types, Lewis lung carcinoma cells (mouse), T241fibrosarcoma (mouse), and Burkitt's lymphoma cells (human), both fromfresh tumor or cell culture after several passages were analysed todetermine the presence of angiostatin transcripts. Northern analysis ofsamples showed an absence of any signal hybridizing with the sequencefrom all samples except that of normal mouse liver RNA showing a singlesignal of approximately 2.4 kb corresponding to mouse plasminogen.Northern analysis of human samples show an absence of any signalhybridizing with human angiostatin sequence from all samples except thatof normal human liver RNA showing a single signal of approximately 2.4kb corresponding to human plasminogen.

Reverse transcription polymerase chain reaction (RT-PCR) analysis showedan absence of any product from all samples probed with mouse angiostatinsequences except that of the normal mouse liver. RT-PCR analysis showedan absence of any product from all human samples probed with humanangiostatin sequences except that of the normal human liver (expectedsize of 1050 bp for mouse and 1134 bp for human).

Thus it appears that mouse angiostatin transcripts (assuming identitywith amino acids 97 to 450 of mouse plasminogen) are not produced by allthe above mouse samples and human angiostatin transcripts (assumingidentity with amino acids 93 to 470 of human plasminogen) are notproduced by the above human samples. The positive signals obtained innormal mouse/human liver is from hybridization with plasminogen.

EXAMPLE 25 Expression of Angiostatin in Yeast

The gene fragment encoding amino acids 93 to 470 of human plasminogenwas cloned into the XhoI/EcoRI site of pHIL-SI (Invitrogen) which allowsthe secreted expression of proteins using the PHO1 secretion signal inthe yeast Pichia pastoris. Similarly, the gene fragment encoding aminoacids 93 to 470 of human plasminogen was cloned into the SnaBI/EcoRIsite of pPIC9 (Invitrogen) which allows the secreted expression ofproteins using the a-factor secretion signal in the yeast Pichiapastoris. The expressed human angiostatin proteins in these systems willhave many advantages over those expressed in E. coli such as proteinprocessing, protein folding and posttranslational modification inclusiveof glycosylation.

Expression of gene in P. pastoris: is described in) Sreekrishna, K. etal. (1988) High level expression of heterologous proteins inmethylotropic yeast Pichia pastoris. J. Basic Microbiol. 29 (4):265-278, and Clare, J. J. et al. (1991) Production of epidermal growthfactor in yeast: High-level secretion using Pichia pastoris strainscontaining multiple gene copies, Gene 105:205-212, both of which arehereby incorporated herein by reference.

EXAMPLE 26 Expression of Angiostatin Proteins in Transgenic Animals andPlants

Transgenic animals such as of the bovine or procine family are createdwhich express the angiostatin gene transcript. The transgenic animalexpress angiostatin protein for example in the milk of these animals.Additionally edible transgenic plants which express the angiostatin genetranscript are constructed.

Constructing transgenic animals that express foreign DNA is described inSmith H. Phytochrome transgenics: functional, ecological andbiotechnical applications, Semin. Cell. Biol. 1994 5(5):315-325, whichis hereby incorporated herein by reference.

EXAMPLE 27 Characterization of Endothelial Cell Proliferation InhibitingAngiostatin Fragments

The following example characterizes the activity of individual andcombinational angiostatin fragments. The data suggests that a functionaldifference exists among individual kringle structures, and potentanti-endothelial, and hence anti-angiogenic, activity can be obtainedfrom such protein fragments of angiostatin.

As used herein, “angiostatin fragment” means a protein derivative ofangiostatin, or plasminogen, having an endothelial cell proliferationinhibiting activity. Angiostatin fragments are useful for treatingangiogenic-mediated diseases or conditions. For example, angiostatinfragments can be used to inhibit or suppress tumor growth. The aminoacid sequence of such an angiostatin fragment, for example, can beselected from a portion of murine plasminogen (SEQ ID NO:1), murineangiostatin (SEQ ID NO:2); human angiostatin (SEQ ID NO:3), Rhesusangiostatin (SEQ ID NO:4), porcine angiostatin (SEQ ID NO:5), and bovineangiostatin (SEQ ID NO:6), unless indicated otherwise by the context inwhich it is used.

As used herein, “kringle 1” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 1, exemplified by, but not limited to that ofmurine kringle 1 (SEQ ID NO:7), human kringle 1 (SEQ ID NO:8), Rhesuskringle 1 (SEQ ID NO:9), porcine kringle 1 (SEQ ID NO:10), and bovinekringle 1 (SEQ ID NO:11), unless indicated otherwise by the context inwhich it is used. Murine kringle 1 (SEQ ID NO:7) corresponds to aminoacid positions 103 to 181 (inclusive) of murine plasminogen of SEQ IDNO:1, and corresponds to amino acid positions 6 to 84 (inclusive) ofmurine angiostatin of SEQ ID NO:2. Human kringle 1 (SEQ ID NO:8), Rhesuskringle 1 (SEQ ID NO:9), porcine kringle 1 (SEQ ID NO:10), and bovinekringle 1 (SEQ ID NO:11) correspond to amino acid positions 6 to 84(inclusive) of angiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, andSEQ ID NO:6, respectively.

As used herein, “kringle 2” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 2, exemplified by, but not limited to that ofmurine kringle 2 (SEQ ID NO:12), human kringle 2 (SEQ ID NO:13), Rhesuskringle 2 (SEQ ID NO:14), porcine kringle 2 (SEQ ID NO:15), and bovinekringle 2 (SEQ ID NO:16), unless indicated otherwise by the context inwhich it is used. Murine kringle 2 (SEQ ID NO:12) corresponds to aminoacid positions 185 to 262 (inclusive) of murine plasminogen of SEQ IDNO:1, and corresponds to amino acid positions 88 to 165 (inclusive) ofmurine angiostatin of SEQ ID NO:2. Human kringle 2 (SEQ ID NO:13),Rhesus kringle 2 (SEQ ID NO:14), porcine kringle 2 (SEQ ID NO:15), andbovine kringle 2 (SEQ ID NO:16) correspond to amino acid positions 88 to165 (inclusive) of angiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,and SEQ ID NO:6, respectively.

As used herein, “kringle 3” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 3, exemplified by, but not limited to that ofmurine kringle 3 (SEQ ID NO:17), human kringle 3 (SEQ ID NO:18), Rhesuskringle 3 (SEQ ID NO:19), porcine kringle 3 (SEQ ID NO:20), and bovinekringle 3 (SEQ ID NO:21). Murine kringle 3 (SEQ ID NO:17) corresponds toamino acid positions 275 to 352 (inclusive) of murine plasminogen of SEQID NO:1, and corresponds to amino acid positions 178 to 255 (inclusive)of murine angiostatin of SEQ ID NO:2. Human kringle 3 (SEQ ID NO:18),Rhesus kringle 3 (SEQ ID NO:19), porcine kringle 3 (SEQ ID NO:20), andbovine kringle 3 (SEQ ID NO:21) correspond to amino acid positions 178to 255 (inclusive) of angiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6, respectively.

As used herein, “kringle 4” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 4, exemplified by, but not limited to that ofmurine kringle 4 (SEQ ID NO:22) and human kringle 4 (SEQ ID NO:23),unless indicated otherwise by the context in which it is used. Murinekringle 4 (SEQ ID NO:22) corresponds to amino acid positions 377 to 454(inclusive) of murine plasminogen of SEQ ID NO:1.

As used herein, “kringle 2-3” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 2-3, exemplified by, but not limited to that ofmurine kringle 2-3 (SEQ ID NO:24), human kringle 2-3 (SEQ ID NO:25),Rhesus kringle 2-3 (SEQ ID NO:26), porcine kringle 2-3 (SEQ ID NO:27),and bovine kringle 2-3 (SEQ ID NO:28), unless indicated otherwise by thecontext in which it is used. Murine kringle 2-3 (SEQ ID NO:24)corresponds to amino acid positions 185 to 352 (inclusive) of murineplasminogen of SEQ ID NO:1, and corresponds to amino acid positions 88to 255 (inclusive) of murine angiostatin of SEQ ID NO:2. Human kringle2-3 (SEQ ID NO:25), Rhesus kringle 2-3 (SEQ ID NO:26), porcine kringle2-3 (SEQ ID NO:27), and bovine kringle 2-3 (SEQ ID NO:28) correspond toamino acid positions 88 to 255 (inclusive) of angiostatin of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

As used herein, “kringle 1-3” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 1-3, exemplified by, but not limited to that ofmurine kringle 1-3 (SEQ ID NO:29), human kringle 1 (SEQ ID NO:30),Rhesus kringle 1-3 (SEQ ID NO:31), porcine kringle 1-3 (SEQ ID NO:32),and bovine kringle 1-3 (SEQ ID NO:33), unless indicated otherwise by thecontext in which it is used. Murine kringle 1-3 (SEQ ID NO:29)corresponds to amino acid positions 103 to 352 (inclusive) of murineplasminogen of SEQ ID NO:1, and corresponds to amino acid positions 6 to255 (inclusive) of murine angiostatin of SEQ ID NO:2. Human kringle 1-3(SEQ ID NO:30), Rhesus kringle 1-3 (SEQ ID NO:31), porcine kringle 1-3(SEQ ID NO:32), and bovine kringle 1-3 (SEQ ID NO:33) correspond toamino acid positions 6 to 255 (inclusive) of angiostatin of SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

As used herein, “kringle 1-2” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 1-2, exemplified by, but not limited to that ofmurine kringle 1-2 (SEQ ID NO:34), human kringle 1-2 (SEQ ID NO:35),Rhesus kringle 1-2 (SEQ ID NO:36), porcine kringle 1-2 (SEQ ID NO:37),and bovine kringle 1-2 (SEQ ID NO:38), unless indicated otherwise by thecontext in which it is used. Murine kringle 1-2 (SEQ ID NO:34)corresponds to amino acid positions 103 to 262 (inclusive) of murineplasminogen of SEQ ID NO:1, and corresponds to amino acid positions 6 to165 (inclusive) of murine angiostatin of SEQ ID NO:2. Human kringle 1-2(SEQ ID NO:35), Rhesus kringle 1-2 (SEQ ID NO:36), porcine kringle 1-2(SEQ ID NO:37), and bovine kringle 1-2 (SEQ ID NO:38) correspond toamino acid positions 6 to 165 (inclusive) of angiostatin of SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

As used herein, “kringle 1-4” means a protein derivative of plasminogenhaving an endothelial cell inhibiting activity or anti-angiogenicactivity, and having an amino acid sequence comprising a sequencehomologous to kringle 1-4, exemplified by, but not limited to that ofmurine kringle 1-4 (SEQ ID NO:39) and human kringle 1-4 (SEQ ID NO:40),unless indicated otherwise by the context in which it is used. Murinekringle 1-4 (SEQ ID NO:39) corresponds to amino acid positions 103 to454 (inclusive) of murine plasminogen of SEQ ID NO:1.

Kringle 1, kringle 2, kringle 3, kringle 4, kringle 2-3, kringle 1-3,kringle 1-2 and kringle 1-4 amino acid sequences are respectivelyhomologous to the specific kringle sequences identified above.Preferably, the amino acid sequences have a degree of homology to thedisclosed sequences of at least 60%, more preferably at least 70%, andmore preferably at least 80%. It should be understood that a variety ofamino acid substitutions, additions, deletions or other modifications tothe above listed fragments may be made to improve or modify theendothelial cell proliferation inhibiting activity or anti-angiogenicactivity of the angiostatin fragments. Such modifications are notintended to exceed the scope and spirit of the claims. For example, toavoid homodimerization by formation of inter-kringle disulfide bridges,the cysteine residues C4 in recombinant human kringle 2 (SEQ ID NO:13)and C42 in recombinant kringle 3 (SEQ ID NO:18) were mutated to serines.Furthermore, it is understood that a variety of amino acidsubstitutions, additions, deletions or other modifications can be madein the above identified angiostatin fragments, which do notsignificantly alter the fragments' endothelial cell proliferationinhibiting activity, and which are, therefore, not intended to exceedthe scope of the claims. By “not significantly alter” is meant that theangiostatin fragment has at least 60%, more preferably at least 70%, andmore preferably at least 80% of the endothelial cell proliferationinhibiting activity compared to that of the closest homologousangiostatin fragment disclosed herein.

Gene Construction and Expression

A PCR-based method was used to generate the cDNA fragments coding forkringle 1 (K1), kringle 2 (K2), kringle 3 (K3), kringle 4 (K4) andkringle 2-3 (K2-3) of human plasminogen (HPg). Recombinant kringle 1(rK1), kringle 2 (rK2), kringle 3 (rK3), kringle 4 (rK4) and kringle 2-3(rK2-3) were expressed in E. coli as previously described (Menhart, N.,Shel, L. C., Kelly, R. F., and Castellino, F. J. (1991) Biochem. 30,1948-1957; Marti, D., Schaller, J., Ochensberger, B., and Rickli, E. E.(1994) Eur. J. Biochem. 219, 455-462; Söhndel, S., Hu, C.-K., Marti, D.,Affolter, M., Schaller, J., Llinas, M., and Rickli, E. E. (1996)Biochem. in press; Rejante, M. R., Byeon, I.-J. L., and Llinas, M.(1991) Biochem. 30, 11081-11092). To avoid homodimerization by formationof inter-kringle disulfide bridges as shown in FIG. 32B, the cysteineresidues C169 and rK2 and C297 in rK3 were mutated to serines as seen inSEQ ID NO.s 13 and 18, at positions 4 and 42, respectively. (Söhndel,S., Hu, C.-K., Marti, D., Affolter, M., Schaller, J., Llinas, M., andRickli, E. E. (1996) Biochem. in press). The rK3 and rK2-3 contained anN-terminal hexa-histidine tag which was used for protein purification(not shown).

Proteolytic Digestion

The fragments of K1-3, K1-4 and K4 were prepared by digestion of Lys-HPg(Abbott Labs) with porcine elastase (Sigma) as previously described(Powell, J. R., and Castellino, F. J. (1983) Biochem. 22, 923-927).Briefly, 1.5 mg elastase was incubated at room temperature with 200 mgof human plasminogen in 50 mM Tris-HCl pH 8.0 overnight with shaking.The reaction was terminated by the addition of diisopropylfluorophosphate (DFP) (Sigma) to a final concentration of 1 mM. Themixture was rocked for an additional 30 minutes at room temperature anddialyzed overnight against 50 mM Tris-HCl, pH 8.0.

Protein Purification

Recombinant K1 was expressed in DH5a E. coli bacterial cells using apSTII plasmid vector. This protein was purified to homogeneity bychromatography using lysine-Sepharose 4B (Pharmacia) and Mono Q (BioRad)columns. E. coli bacterial cells (strain HB101) expressing rK2 and rK3were grown to an OD₆₀₀ of approximately 0.8 at 3° C. in 2×YT mediumcontaining 100 mg/ml ampicillin and 25 mg/ml kanamycin. IPTG(isopropyl-b-D-thiogalactopyranoside) was added to a final concentrationof 1 mM and cells were grown for an additional 4.5 hours at 37° C. toinduce the production of recombinant proteins. The cells were harvestedby centrifugation and the pellets were stored at −80° C. The thawed celllysates were re-suspended in the extraction buffer (6 M guanidinehydrochloride in 0.1M sodium phosphate, pH 8.0). The suspension wascentrifuged at 15,000×g for 30 minutes and b-mercaptoethanol was addedto the supernatant at a final concentration of 10 mM. The supernatantwas then loaded on a Ni²⁺-NTA agarose column (1.5 cm×5 cm)pre-equilibrated with the extraction buffer. The column was washedsuccessively with extraction buffer at pH 8.0 and pH 6.3, respectively.Recombinant K2 and K3 were eluted with extraction buffer at pH 50.

The proteolytically cleaved fragments of K1-3, K1-4 and K4 were purifiedusing a lysine-Sepharose 4B column (2.5 cm×15 cm) equilibrated with 50mM Tris-HCl, pH 8.0 until an absorbance at 180 nm reached 0.005. Theabsorbed kringle fragments were eluted with Tris buffer containing 200mM ε-aminocaproic acid, pH 8.0. The eluted samples were the dialyzedovernight against 20 mM Tris-HCl, pH 5.0, and were applied to a BioRadMono-S column equilibrated with the same buffer. The fragments of K4,K1-3 and K1-4 were eluted with 0-20%, 20-50% and 50-70% step-gradientsof 20 mM phosphate/1 M KCl, pH 5.0. Most K1-3 and K1-4 fragments wereeluted from the column with 0.5 M KCl as determined by SDS-PAGE. Allfractions were dialyzed overnight against 20 mM Tris-HCl, pH 8.0. Afterdialysis, K1-3 and K1-4 fragments were further purified using aheparin-Sepharose column (5 cm×10 cm) (Sigma) pre-equilibrated with 20mM Tris-HCl buffer, pH 8.0. The K1-3 fragment was eluted with 350 mM KCland K1-4 was recovered from the flow-through fraction. The purifiedkringle fragments were analyzed on SDS-gels follows by silver-staining,by Western immunoblotting analysis with anti-human K4 and K1-3polyclonal antibodies, and by amino-terminal sequencing analysis.

In Vitro Re-Folding

The re-folding of rK2, rK3 and rK2-3 was performed according to astandard protocol (Cleary, S., Mulkerrin, M. G., and Kelley, R. R.(1989) Biochem. 28, 1884-1891). The purified proteins were adjusted topH 8.0 and dithiothreitol (DTT) was added to a final concentration of 5mM. After an overnight incubation, the solution was diluted with 4volumes of 50 mM Tris-HCl, pH 8.0, containing 1.25 mM reducedglutathione. After 1 hour of incubation, oxidized glutathione was addedto a final concentration of 1.25 mM and incubated for 6 hours at 4° C.The renatured protein was dialyzed initially against H₂O for 2 days andfor an additional two days against 50 mM phosphate-buffered saline, pH8.0. The solution was then loaded onto a lysine-Bio-Gel column (2 cm×13cm) equilibrated with the same phosphate-buffered saline. The column waswashed with phosphate-buffered saline and protein was eluted with aphosphate buffer containing 50 mM 6-AHA (6-aminohexanoic acid).Reverse-phase HPLC was performed on an Aquapore Butyl column (2.1×100mm, widepore 30 nm, 7 mm, Applied Biosystems) and a Hewlett Packardliquid chromatography was used with acetonitrile gradients.

Reduction and Alkylation

The reduction and alkylation of kringle fragments were performedaccording to a standard protocol (Cao, Y., and Pettersson, R. F., (1990)Growth Factors 3, 1013). Approximately 20-80 mg of purified proteins in300-500 ml DME medium in the absence of serum were incubated at roomtemperature with 15 ml of 0.5 M DTI for 15 minutes. After incubation, 30ml of 0.5 M iodoacetamide was added to the reaction. The proteinsolution was dialyzed at 4° C. overnight initially against 20 volumes ofDMEM. The solution was further dialyzed at 4° C. for an additional 4hours against 20 volumes of fresh DMEM. After dialysis, the samples wereanalyzed on a SDS-gel and assayed for their inhibitory activities onendothelial cell proliferation.

Endothelial Proliferation Assay

Bovine capillary endothelial (BCE) cells were isolated as previouslydescribed (Folkman, J., Haudenschild, C. C., and Zetter, B. R. (1979)Proc. Natl. Acad. Sci USA. 76, 5217-5121) and maintained in DMEMsupplemented with 10% heat-inactivated bovine calf serum (BCS),antibiotics, and 3 ng/ml recombinant human bFGF (Scios Nova,Mountainview, Calif.). Monolayers of BCE cells growing in 6-well plateswere dispersed in a 0.05% trypsin solution. Cells were re-suspended withDMEM containing 10% BCS. Approximately 12,500 cells in 0.5 ml were addedto each well of gelatinized 24-well tissue culture plates and incubatedat 37° C. (in 10% CO₂) for 24 hours. The medium was replaced with 500 mlof fresh DMEM containing 5% BCS and samples of individual orcombinatorial kringle fragments in triplicates were added to each well.After 30 minutes of incubation, bFGF was added to a final concentrationof 1 ng/ml. After 72 hours of incubation, cells were trypsinized,re-suspended in Hematall (Fisher Scientific, Pittsburgh, Pa.) andcounted with a Coulter counter.

Purification and Characterization of Kringle Fragment of HumanPlasminogen

The cDNA fragments coding for individual kringles (K1, K2, K3, and K4)and kringles 2-3 (K2-3) of human plasminogen were amplified by aPCR-based method (FIG. 28). The PCR-amplified cDNA fragments were clonedinto a bacterial expression vector. Recombinant proteins expressed fromEscherichia coli were refolded in vitro and were purified to >98%homogeneity using HPLC-coupled chromatography (FIG. 29). Under reducingconditions, recombinant K2, K3 and K4 migrated with molecular weights of12-13 kDa (FIG. 29A, lanes 2-4), corresponding to the predictedmolecular weights of each kringle fragment. Recombinant K1 migratingwith a higher molecular weight of 17 kDa was identified by SDS-gelelectrophoresis. The fragments of K1-4 and K1-3 were obtained byproteolytic digestion of human Lys-plasminogen (Lys-HPg) with elastaseas previously described (Powell, J. R., and Castellino, F. J. (1983)Biochem. 22, 923-927; Brockway, W. J., and Castellino, F. J. (1972)Arch. Biochem. Biophys). These two fragments (FIG. 29B, lanes 1 and 2)with predicted molecular weights of 43 kDa and 35 kDa, respectively,were also purified to homogeneity. N-terminal amino acid sequenceanalysis of the purified fragments yielded an identical sequence,-YLSE-, followed by SEQ ID NO:30 and SEQ ID NO:40, for K1-3 and K1-4,respectively. The N-terminal sequence for K4 produced -VVQD- withapproximately 20% -VQD-, followed by SEQ ID NO:23, each of which ispredicted from the expected sequence beginning with Valine¹⁷⁶ andValine¹⁷⁷ of human angiostatin (SEQ ID NO: 3).

Anti-Endothelial Cell Proliferative Activity of Individual Kringles

Individual recombinant kringle fragments of angiostatin were assayed forthe inhibitory activities on bovine capillary endothelial (BCE) cellgrowth stimulated by bFGF. As shown in FIG. 30A, rK1 inhibited BCE cellproliferation in a dose-dependent fashion. The concentration of rK1required to reach 50% inhibition (ED₅₀) was about 320 nM (Table 4). Incontrast, rK4 exhibited little or no inhibitory effect on endothelialcell proliferation. Recombinant K2 and rK3, two non-lysine bindingkringle fragments, also produced a dose-dependent inhibition ofendothelial cell proliferation (FIG. 30B). However, the inhibitorypotency of rK2 was substantially lower than rK1 and rK3 (ED₅₀=460) (FIG.30 and Table 4). No cytotoxicity or distinct morphology associated withapoptotic endothelial cells such as rounding, detachment, andfragmentation of cells could be detected, even after incubation with ahigh concentration of these kringle fragments. These data suggest thatthe anti-endothelial growth activity of angiostatin may be shared byfragments of K1, K2 and K3, and lesser so by K4.

TABLE 4 Inhibitory activity on capillary endothelial cell proliferation.Fragments ED₅₀ (nM) Kringle 1 320 Kringle 2 — Kringle 3 460 Kringle 4 —Kringle 2-3 — Kringle 1-3 70 Kringle 1-4 (Angiostatin) 135

Anti-Endothelial Cell Proliferative Activity of K1-3 and K1-4 Fragments

To evaluate the anti-endothelial cell proliferative effect of combinedkringle fragments, purified proteolytic fragments of human K1-4, K1-3and rK2-3 were assayed on BCE cells. In agreement with previous findings(O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A.,Moses, J., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994)Cell 79, 315-328), BCE cell proliferation, as shown in FIG. 31, wassignificantly inhibited by angiostatin-like fragment K1-4 (ED₅₀=135 nM)(Table 4). An increase of anti-endothelial growth activity was obtainedwith K1-3 fragment (ED₅₀=70 nM) (Table 4). The inhibition of endothelialcell proliferation occurred in a dose-dependent manner. These resultsindicate that removal of K4 from angiostatin potentiatesanti-endothelial growth activity.

Additive Inhibition by rK2 and rK3

The fragment of rK2-3 displayed only weak inhibitory activity which wassimilar to that of rK2 alone (FIG. 31). However, both rK2 and rK3inhibited endothelial cell proliferation (FIG. 30B). This findingsuggested that the inhibitory effect of K3 was hidden in the structureof K2-3. Previous structural studies showed that an inter-kringledisulfide bond was present between K2 (cysteine¹⁶⁹) and K3 (cysteine²⁹⁷)of human plasminogen, corresponding to cysteine⁹¹ and cysteine²¹⁹ of SEQID NO: 3 (Söhndel, S., Hu, C.-K., Marti, D., Affolter, M., Schaller, J.,Llinas, M., and Rickli, E. E. (1996) Biochem. in press) See FIG. 32B.The inhibitory effect of rK2 and rK3 in combination was tested.Interestingly, an additive inhibition was seen when individual rK2 andrK3 fragments were added together to BCE cells. See FIG. 32A. Theseresults imply that it is preferable to open the interdisulfide bridgebetween K2 and K3 in order to obtain the maximal inhibitory effect ofK2-3.

Appropriate Folding of Kringle Structures is Required for theAnti-Endothelial Activity of Angiostatin

To study whether the folding of kringle structures is required for theanti-endothelial proliferation activity, native angiostatin was reducedwith DTT and assayed on bovine capillary endothelial cells. Afterreduction, angiostatin was further alkylated with iodoacetamide andanalyzed by SDS gel electrophoresis. As shown in FIG. 34A, theDTT-treated protein migrated at a higher position with molecular weightof about 42 kDa (lane 2) as compared to the native angiostatin withmolecular weight of 33 kDa (lane 1), suggesting that angiostatin wascompletely reduced. The anti-proliferation activity of angiostatin waslargely abolished after reduction (FIG. 34B). From these results, weconclude that the correct folding of angiostatin through theintra-kringle disulfide bonds is preferable to maintain its potenteffect on inhibition of endothelial cell proliferation.

Amino acid sequence alignment of the kringle domains of humanplasminogen shows that K1, K2, K3 and K4 display identical grossarchitecture and remarkable sequence homology (56-82% identify) as seenin FIG. 35. Among these structures, the high-affinity lysine bindingkringle, K1, is the most potent inhibitory segment of endothelial cellproliferation. Of interest, the intermediate-affinity lysine bindingfragment, K4, lacks inhibitory activity. These data suggest that thelysine binding site of the kringle structures may not be directlyinvolved in the inhibitory activity. The amino acid conservation andfunctional divergence of these kringle structures provide an idealsystem to study the role mutations caused by DNA replication duringevolution. Similar divergent activities relative to the regulation ofangiogenesis exhibited by a group of structurally related proteins arealso found in the —C—X—C— chemokine and prolactin-growth hormonefamilies (Maione, T. E., Gray, G. S., Petro, A. J., Hunt, A. L., andDonner, S. I. (1990) Science 247, 77-79.; Koch, A. E., Polverini, P. J.,Kunkel, S. L., Harlow, L. A., DiPietro, L. A., Elner, V. M., Elner, S.J., and Strieter, R. M. (1992) Science 258, 1798-1801.; Cao, Y., Chen,C., Weatherbee, J. A., Tsang, M., and Folkman, J. (1995) J. Exp. Med.182, 2069-2077.; Strieter, R. M., Polverini, P. J., Arenberg, D. A., andKunkel, S. L. (1995) Shock 4, 155-160.; Jackson, D., Volpert, O. V.,Bouck, N., and Linzer, D. I. H. (1994) Science 266, 1581-1584).

Further sequence analysis reveals that K4 contains two positivelycharged lysine residues adjacent to cysteines 22 and 78 (FIG. 35). ¹Hnuclear magnetic resonance (NMR) analysis shows that these 4 lysines,together with lysine 57, form the core of a positively charged domain inK4 (Llinas M, unpublished data), whereas other kringle structures lacksuch a positively charged domain. Whether this lysine-enriched domaincontributes to the loss of inhibitory activity of kringle 4 of humanplasminogen remains to be studied. K4 was previously reported tostimulate proliferation of other cell types and to increase the releaseof intracellular calcium (Donate, L. E., Gherardi, E., Srinivasan, N.,Sowdhamini, R., Aporicio, S., and Blundell, T. L. (1994) Prot. Sci. 3,2378-2394). The fact that removal of K4 from angiostatin potentiates itsinhibitory activity on endothelial cells suggests that this structuremay prevent some of the inhibitory effect of K1-3.

The mechanism underlying how angiostatin and its related kringlefragments specifically inhibit endothelial cell growth remainsuncharacterized. It is not yet clear whether the inhibition is mediatedby a receptor that is specifically expressed in proliferatingendothelial cells, or if angiostatin is internalized by endothelialcells and subsequently inhibits cell proliferation. Alternatively,angiostatin may interact with an endothelial cell adhesion receptor suchas integrin a_(v)b₃, blocking integrin-mediated angiogenesis (Brooks, P.C., Montgomery, A. M., Rosenfeld, M., Reisfeld R. A., Hu, T. Klier, G.,and Cheresh, D. A. (1994) Cell 79, 1157-1164). Of interest, Friedlanderet. al. (Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M.,Varner, J. A., and Cheresh, D. A. (1995) 270, 1502) reported recentlythat in vivo angiogenesis in cornea or chorioallantoic membrane models(induced by bFGF and by tumor necrosis factor) was a_(v)b₃ integrindependent. However, angiogenesis stimulated by VEGF, transforming growthfactor a, or phorbol esters was dependent on a_(v)b₅. Antibodies to theindividual integrins specifically blocked one of these pathways, and acyclic protein antagonist of both integrins blocked angiogenesis inducedby each cytokine (Friedlander, M., Brooks, P. C., Shaffer, R. W.,Kincaid, C. M., Varner, J. A., and Cheresh, D. A. (1995) 270, 1502).Because bFGF- and VEGF-induced angiogenesis are inhibited byangiostatin, it may block a common pathway for these integrin-mediatedangiogenesis.

An increasing number of endogenous angiogenesis inhibitors have beenidentified in the last few decades (Folkman, J. (1995) N. Engl. J. Med.333, 1757-1763). Of the nine characterized endothelial cell suppressors,several inhibitors are proteolytic fragments. For example, the 16 kDaN-terminal fragment of human prolactin inhibits endothelial cellproliferation and blocks angiogenesis in vivo (Clapp, C., Martial, J.A., Guzman, R. C., Rentierdelrue, F., and Weiner, R. I. (1993)Endorinology 133, 1292-1299). In a recent paper, D'Angelo et. al.reported that the antiangiogenic 16 kDa N-terminal fragment inhibitedthe activation of mitogen-activated protein kinase (MAPK) by VEGF andbFGF in capillary endothelial cells (D'Angelo, G., Struman, I., Martial,J., and Weiner, R. (1995) Proc. Natl. Acad. Sci. 92, 6374-6378). Similarto angiostatin, the intact parental molecule of prolactin does notinhibit endothelial cell proliferation nor is it an angiogenesisinhibitor. Platelet factor 4 (PF-4) inhibits angiogenesis at highconcentrations (Maione, T. E., Gray, G. S., Petro, A. J., Hunt, A. L.,and Donner, S. I. (1990) Science 247, 77-79; Cao, Y., Chen, C.,Weatherbee, J. A., Tsang, M., and Folkman, J. (1995) J. Exp. Med. 182,2069-2077). However, the N-terminally truncated proteolytically cleavedPF-4 fragment exhibits a 30- to 50-fold increase in itsanti-proliferative activity over the intact PF-4 molecule (Gupta, S. K.,Hassel, T., and Singh, J. P. (1995) Proc. Natl. Acad. Sci. 92,7799-7803). Smaller protein fragments of fibronectin, murine epidermalgrowth factor, and thrombospondin have also been shown to specificallyinhibit endothelial cell growth (Homandberg, G. A., Williams, J. E.,Grant, D., Schumacher, B., and Eisenstein, R. (1985) Am. J. Pathol. 120,327-332; Nelson, J., Allen, W. E., Scott, W. N., Bailie, J. R., Walker,B., McFerran, N. V., and Wilson, D. J. (1995) Cancer Res. 55, 3772-3776;Tolsma, S. S., Volpert, O. V., Good, D. J., Frazer, W. A., Polverini, P.J., and Bouck, N. (1993) J. Cell Biol. 122, 497-511). Proteolyticprocessing of a large protein may change the conformational structure ofthe original molecule or expose new epitopes that are antiangiogenic.Thus, protease(s) may play a critical role in the regulation ofangiogenesis. To date, little is known about the regulation of theseprotease activities in vivo.

The data also show that the disulfide bond mediated folding of thekringle structures in angiostatin is preferable to maintain itsinhibitory activity on endothelial cell growth. Kringle structuresanalogous to those in plasminogen are also found in a variety of otherproteins. For example, apolipoprotein (a) has as many as 37 repeats ofplasminogen kringle 4 (McLean, J. W., Tomlinson, J. E., Kuang, W.-J.,Eaton, D. L., Chen, E. Y., Fless, G. M., Scanu, A. M., and Lawn, R. M.(1987) Nature 330, 132-137). The amino terminal portion of prothrombinalso contains two kringles that are homologous to those of plasminogen(Walz, D. A., Hewett-Emmett, D., and Seegers, W. H. (1977) Proc. Natl.Acad. Sci. 74, 1969-1973). Urokinase has been shown to possess a kringlestructure that shares extensive homology with plasminogen (Gunzler, W.A., J., S. G., Otting, F., Kim, S.-M. A., Frankus, E., and Flohe, L.(1982) Hoppe-Seyler's A. Physiol. Chem. 363, 1155-1165). In addition,surfactant protein B and hepatocyte growth factor (HGF), also carrykringle structures (Johansson, J., Curstedt, T., and Jörnvall., H.(1991) Biochem. 30, 6917-6921; Lukker, N. A., Presta, L. G., andGodowski, P. J. (1994) Prot. Engin. 7, 895-903).

EXAMPLE 28 Suppression of Metastases and of Endothelial CellProliferation by Angiostatin Fragments

The following example characterizes the activity of additionalangiostatin fragments. The data suggests that potent anti-endothelialand tumor suppressive activity can be obtained from such proteinfragments of angiostatin.

As used herein, “kringle 1-4BKLS” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity, and havingan amino acid sequence comprising a sequence homologous to kringle1-4BKLS, exemplified by, but not limited to that of murine kringle1-4BKLS (SEQ ID NO:41), and human kringle 1-4BKLS (SEQ ID NO:42), unlessindicated otherwise by the context in which it is used. Murine kringle1-4BKLS (SEQ ID NO:41) corresponds to amino acid positions 93 to 470(inclusive) of murine plasminogen of SEQ ID NO:1. This exampledemonstrates that an “angiostatin fragment” can be a plasminogenfragment and encompass an amino acid sequence larger than theangiostatin presented in SEQ ID NO:3, for example, and still havetherapeutic endothelial cell proliferation inhibiting activity oranti-angiogenic activity.

A kringle 1-4BLKS amino acid sequence is homologous to the specifickringle 1-4BLKS sequences identified above. Preferably, the amino acidsequences have a degree of homology to the disclosed sequences of atleast 60%, more preferably at least 70%, and more preferably at least80%. It should be understood that a variety of amino acid substitutions,deletions and other modifications to the above listed fragments may bemade to improve or modify the endothelial cell inhibiting activity ofthe fragments. Such modifications are not intended to exceed the scopeand spirit of the claims. Furthermore, it is understood that a varietyof silent amino acid substitutions, additions, or deletions can be madein the above identified kringle fragments, which do not significantlyalter the fragments' endothelial cell inhibiting activity, and whichare, therefore, not intended to exceed the scope of the claims.

Cloning of Angiostatin in Pichia pastoris

Sequences encoding angiostatin were amplified by PCR using Ventpolymerase (New England Biolabs) and primers #154(5′-ATCGCTCGAGCGTTATTTGAAAAGAAAGTG-3′) (SEQ ID NO:43) and #151(5′-ATCGGAATTCAAGCAGGACAACAGGCGG-3′) (SEQ ID NO:44) containing linkersXhol and Eco Rl respectively and using the plasmid pTrcHis/HAs astemplate. This plasmid contained sequences encoding amino acids 93 to470 of human plasminogen (SEQ ID NO:42) for cloning into the Xho I/ECoR1 site of pHIL-S1 expression vector using the P. pastoris nativesecretion signal PHO 1. This same sequence was amplified in the samemanner using primers #156 (5′-ATCGTACGTATTATTTGAAAAGAAAGTG-3′) (SEQ IDNO:45) and #151 containing linkers Sna Bl and Eco RI respectively, forcloning into the Sna Bl/ECo Rl site of expression vector pPlC9 with thealpha-factor secretory signal. The products of the amplifications weregel purified, linkers were digested with the appropriate enzymes, andagain purified using gene-clean (Bio 101). These gene fragments wereligated into the appropriate vectors. Resultant clones were selected andplasmid preparations of clones were obtained and linearized to generateHis⁺ Mut^(s) and His⁺ Mut⁺ recombinant strains when transformed into P.pastoris host strain GS115. Integration was confirmed by PCR.

Both His⁺ and His⁺ Mut⁺ recombinants were induced with methanol andscreened for high expression of angiostatin using Coomassie stainedSDS-PAGE gels and immunoblots using mouse monoclonal antibody againstkringles 1 to 3 (Castellino, Enzyme Research Laboratories, Inc., SouthBend, Ind.). From these, a GS115 transformed P. pastoris clonepHIL-S1/HAs18 was selected and phenotypically characterized as His⁺Mut^(s).

Expression of PHIL-S1/HAs18

Expression of angiostatin from pHIL-S1/HAs18 was typical for a His⁺Mut^(s) clone. At induction in baffled shake flasks, 1 L of OD₆₀₀ cellswere cultured in 150 ml of buffered metanol complex medium containing 1%yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34%yeast nitrogen base with ammonium sulfate, 0.00004% biotin and 0.5%methanol, in a 1 L baffled flask. Cells were constantly shaken at 30°C., 250 rpm. Methanol was batch fed at 24 hour intervals by addition ofabsolute methanol to a final of 0.5%. After 120 hours cells were spun at5,000 rpm for 10 minutes, and supernatants were stored at −70° C. untilused.

Purification of Angiostatin from P. pastoris Fermentation Broth byLysine-Sepharose Chromatography

All procedures are carried out at 4° C. Crude fermentation broth,typically 200 ml, containing angiostatin was clarified by centrifugationat 14,000×g and concentrated by Centriprep 30 (amicon) 30 kDa molecularweight cutoff membrane to approximately one-fourth the original volume.One volume of 50 mM phosphate buffer, pH 7.5, was added to theconcentrated sample which was again concentrated by Centriprep toone-fourth the original sample volume. The sample was again dilutedvolume:volume with 50 mM sodium phosphate buffer, pH 7.5. 60 glysine-sepharose 4B (Pharmacia) was resuspended in 500 ml ice-cold 50 mMphosphate buffer, pH 7.5 and used to pack a 48×100 mm column (−180 mlpacked volume). The column was washed overnight with 7.5 column volumes(CV) of 50 mM sodium phosphate buffer, pH 7.5, at a flow rate of 1.5ml/min. The sample was pumped onto the column at a flow rate of 1.5ml/min and the column washed with 1.5 CV of 50 mM sodium phosphate, pH7.5, at a flow rate of 3 ml/min. The column was then washed with 1.5 CVphosphate-buffered saline, pH 7.4, at a flow rate of 3 ml/min:angiostatin was then eluted with 0.2 M ε-amino-n-caproic acid, pH 7.4 ata flow rate of 3 ml/min. Fractions containing significant absorbancewere pooled and dialyzed for 24-48 hours against deionized water andlyophilized. A typical recovery from a 100 mg total protein load is 10mg angiostatin. Columns were regenerated using 5 column volumes of 50 mMsodium phosphate/1 M NaCl, pH 7.5.

Bovine Capillary Endothelial Cell Proliferation Assay

Bovine capillary endothelial cells were obtained as previouslydescribed. The cells are maintained in DMEM containing 3 mg/ml ofrecombinant human bFGF (Scios Nova, Mountainview, Calif.), supplementedwith 10% heat-inactivated bovine calf serum, 100 U/ml penicillin, 100mg/ml streptomycin, and 0.25 mg/ml fungizone (BioWhittaker) in 75 cm²cell-culture flasks. The assay was performed as described previously.

Animal Studies

Six to eight week old male C57BI/6J mice (Jackson Laboratories) wereinoculated subcutaneously with murine Lewis lung carcinoma-lowmetastatic (LLC-LM) line (1×10⁶ cells/injection). Approximately 14 daysafter implantation, when primary tumor reached 1.5 cm³, animals wereanaesthetized with methoxyflurane and primary tumors were surgicallyexcised. The incision site was closed with simple interrupted sutures.Half the animals in this group received a loading dose (3 mg/kg by thesubcutaneous route) of recombinant or plasminogen derived angiostatinsubcutaneously immediately after surgery, followed by daily inoculationsof 1.5 mg/kg for 14 days. A control group of mice received an equalvolume of PBS every day for 14 days following surgery. All mice weresacrificed 14 days after primary tumor removal (28 days after tumorimplantation), lungs were removed and weighed, and surface metastaseswere counted with stereomicroscope.

Characteristics of Recombinant Human Angiostatin Fragments

A gene fragment encoding human angiostatin including kringles 1 to 4 ofhuman plasminogen that contains a total of 26 cysteines, was expressedin Pichia pastoris, the methylotropic yeast. P. pastoris expressedangiostatin binds lysine sepharose and can be specifically eluted byε-amino caproic acid. This demonstrates that fully functional epsilonamino caproic acid-binding kringle(s), which are physical properties ofkringle 1 and 4 of plasminogen (Sottrup-Jensen, L. et al., Progress inChemical Fibrinolysis and Thrombolysis, Vol. 3 (1978) Ravens Press, N.Y.p. 191), can be expressed and secreted by P. pastoris and purified bytechniques that do not require refolding (FIGS. 36A and B). Expressedangiostatin from P. pastoris as well as angiostatin purified by elastasecleavage of plasminogen were recognized by a conformationally dependentmonoclonal antibody against kringle 1 to 3 (Castellino, Enzyme ResearchLaboratories, Inc., South Bend, Ind.) (FIG. 36B). This antibody fails torecognize reduced forms of plasminogen or angiostatin.

P. pastoris expressed angiostatin is seen as a doublet that migrates at49 kDa and 51.5 kDa on denatured unreduced SDS-PAGE Coomassie stainedgels. P. pastoris expressed proteins are post-translationally modifiedwith the majority of N-linked glycosylation of the high-mannose type andinsignificant O-linked glycosylation. To evaluate the possibility ofglycosylation in P. pastoris expressed angiostatin, we digested therecombinant angiostatin with endoglycosidase H specific for high mannosestructures, causing the 51.5 kDa band to migrate identically with theband at 49 kDa (FIGS. 37A and B). O-glycanase digestion with priorneuraminidase treatment to remove sialic acid residues, did not changethe pattern of migration of the doublet (data not shown). These resultsindicate that P. pastoris expressed angiostatin in two forms: (1) withan N-linked complex chain probably of the structure:

and (2) without any glycosylation.

Inhibition of Bovine Capillary Endothelial Cells In Vitro

To determine if recombinantly expressed angiostatin had the potentialfor antiangiogenic activity, BCEs were cultured in the presence of bFGFto determine if the addition of purified recombinant angiostatin wouldinhibit the proliferation of BCEs. Purified P. pastoris-expressedangiostatin inhibited the bFGF-driven proliferation of bovineendothelial cells in vitro (FIG. 38B) in a dose dependent manner (FIG.38C). At 1 ug/ml of recombinant angiostatin, inhibition was 80%. The 50%inhibition was equivalent to that obtained with angiostatin derived fromelastase cleavage of human plasminogen.

Suppression of Metastases In Vivo

The transplantable murine LLC (LM) line from which angiostatin was firstidentified was used. When implanted subcutaneously in syngenic C57B1/6Jmice, these tumors grow rapidly, producing >1.5 cm³ tumors within 14days. Following primary tumor resection, the micrometastases in thelungs grow exponentially, to completely cover the surface of the lung.These metastases are highly vascularized by day 14 after primary tumorresection. If the primary tumor is left on, the micrometastases remaindormant and are not macroscopically visible. Recombinant angiostatin wasadministered systemically to mice following primary tumor resection totest the suppression of the growth of metastases. P. pastoris expressedangiostatin administered systemically at 30 ug/mouse/day inhibited thegrowth of metastases as quantitated by scoring of surface metastases(FIG. 39A) and total lung weight (FIG. 39B). The weights of lungs ofmice that had primary tumors resected and that received daily doses ofrecombinant angiostatin or angiostatin obtained from elastase cleavageof plasminogen were of comparable to those of normal mice (190 to 200mg). Lungs of mice that had their primary tumors resected andsubsequently treated with daily doses of recombinant angiostatin werepink with minimal numbers of unvascularized micrometastases (FIG. 40).In contrast, the mice treated with saline after primary tumor resectionhad lungs covered with vascularized metastases (FIG. 41). Also ofnotable importance was an absence of systemic or local toxicity causedby P. pastoris expressed angiostatin at the dosage and regimen used inthis study. There was no evidence of inflammation or bleeding in alltreated mice.

Angiostatin protein expressed by P. pastoris possesses two importantphysical characteristics of the natural protein: (1) it is recognized bya conformationally dependent monoclonal antibody raised against kringle1 to 3 of human plasminogen (FIG. 36B) and (2) it binds lysine (FIGS.36A and B). These properties indicated that the recombinant angiostatinprotein was expressed with a conformation that mimics the nativemolecule. P. pastoris expressed angiostatin protein inhibits theproliferation of bovine capillary endothelial cells stimulated by bFGFin vitro (FIG. 38). when administered systemically, the recombinantangiostatin maintained the otherwise lethal metastatic Lewis lungcarcinoma in a suppressed state (FIGS. 39A and B and FIG. 40).

Preliminary data shows the absence of a detectable transcript forangiostatin in Lewis lung tumors freshly resected from mice or in LLCcells after 4 passages in in vitro culture. Plasminogen, produced by theliver, is maintained in circulation at a stable plasma concentration of1.6±0.2 μM. It is possible that LLC-LM tumors produce an enzyme thatcleaves plasminogen, bound or in circulation, to produce angiostatin.Alternatively inflammatory cells attracted to the tumor site couldproduce such an enzyme.

It is intriguing that both P. pastoris as well as native humanplasminogen is produced in a glycosylated and a non-glycosylated form.In the case of human plasminogen, a single transcript for a single genecan produce both forms. The molecular mechanism of differentialpost-translational modifications of human plasminogen, as well as thatseen in TPA are unknown.

Angiostatin is highly expressed by P. pastoris. Supernatants contain 100mg/L of the protein. Therefore, the quantities required for clinicaltrials should be straightforward to produce and purify using standardtechnology well-known to those skilled in the art. The development ofthis expression system, and the demonstration of the in vitro and invivo activity of purified recombinant angiostatin against metastasesprovided the foundation for assessment of the capacity of thesefragments to inhibit tumor growth and prolong life in cancer patientsand others suffering from angiogenic-mediated disease.

It should be understood that the foregoing relates only to preferredembodiments of the present invention, and that numerous modifications oralterations may be made therein without departing from the spirit andthe scope of the invention as set forth in the appended claims.

1-63. (canceled)
 64. An isolated nucleic acid molecule consisting of anucleotide sequence encoding a fragment of plasminogen, wherein thefragment of plasminogen consists of a kringle 2-3 fragment, a kringle1-3 fragment, a kringle 1-2 fragment, a kringle 4 fragment, a kringle1-4 fragment, or a kringle 1-4BKLS fragment.
 65. A vector comprising anucleotide sequence encoding a fragment of plasminogen, wherein thefragment of plasminogen consists of a kringle 2-3 fragment, a kringle1-3 fragment, a kringle 1-2 fragment, a kringle 4 fragment, a kringle1-4 fragment, or a kringle 1-4BKLS fragment, and when the vector isexpressed, the fragment of plasminogen is produced.
 66. A method ofmodulating angiogenesis in an animal or a human comprising administeringto the animal or the human a composition comprising a nucleotidesequence encoding a fragment of plasminogen, wherein the fragment ofplasminogen consists of a kringle 2-3 fragment, a kringle 1-3 fragment,a kringle 1-2 fragment, a kringle 4 fragment, a kringle 1-4 fragment, ora kringle 1-4BKLS fragment, and when the vector is expressed, thefragment of plasminogen is produced.